Polynucleotide agents targeting hydroxyacid oxidase (glycolate oxidase, hao1) and methods of use thereof

ABSTRACT

The invention relates to polynucleotide agents targeting an hydroxyacid oxidase (HAO1) gene, and methods of using such polynucleotide agents to inhibit expression of HAO1 and to treat subjects having an HAO1-associated disease, e.g., hyperoxaluria.

RELATED APPLICATIONS

This application is a 35 § U.S.C. 111(a) continuation application whichclaims the benefit of priority to PCT/US2016/037563, filed on Jun. 15,2016, which claims the benefit of priority to U.S. Provisional PatentApplication No. 62/181,602, filed on Jun. 18, 2015. The entire contentsof each of the aforementioned priority applications are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 22, 2017, isnamed 121301_03602_SL.txt and is 149,653 bytes in size.

BACKGROUND OF THE INVENTION

Oxalate (C₂O₄ ²⁻) is the salt-forming ion of oxalic acid (C₂H₂O₄) thatis widely distributed in both plants and animals. It is an unavoidablecomponent of the human diet and a ubiquitous component of plants andplant-derived foods. Oxalate can also be synthesized endogenously viathe metabolic pathway that occurs primarily in the liver. Glyoxylate isan immediate precursor to oxalate and is derived from the oxidation ofglycolate by the enzyme glycolate oxidase (GO), also known, and referredto herein, as hydroxyacid oxidase (HAO1), or by catabolism ofhydroxyproline, a component of collagen. Transamination of glyoxylatewith alanine by the enzyme alanine/glyoxylate aminotransferase (AGT)results in the formation of pyruvate and glycine. Excess glyoxylate willbe converted to oxalate by glycolate oxidase or lactate dehydrogenase.The endogenous pathway for oxalate metabolism is illustrated in FIG. 1.

Oxalic acid may form oxalate salts with various cations, such as sodium,potassium, magnesium, and calcium. Although sodium oxalate, potassiumoxalate, and magnesium oxalate are water soluble, calcium oxalate (CaOx)is nearly insoluble. Excretion of oxalate occurs primarily by thekidneys via glomerular filtration and tubular secretion.

Hyperoxaluria is a condition characterized by increased urinaryexcretion of oxalate. As oxalate can bind with calcium in the kidney,hyperoxaluria can lead to urinary CaOx supersaturation, resulting in theformation and deposition CaOx crystals in renal tissue. These CaOxcrystals may contribute to the formation of diffuse renal calcifications(nephrocalcinosis) and stones (nephrolithiasis). Moreover, when theinnate renal defense mechanisms are suppressed, injury and progressiveinflammation caused by these CaOx crystals, together with secondarycomplications such as tubular obstruction, may lead to decreased renalfunction and in severe cases even to end-stage renal failure.Furthermore, systemic deposition of CaOx (systemic oxalosis) may occurin extrarenal tissues, which can lead to early death if left untreated.

Hyperoxaluria can be generally divided into two categories: primary andsecondary hyperoxaluria. Primary hyperoxaluria is the result ofinherited enzyme deficiencies leading to increased endogenous oxalatesynthesis. Secondary hyperoxaluria results from conditions underlyingincreased intestinal oxalate absorption, such as (1) a high-oxalatediet, (2) fat malabsorption (enteric hyperoxaluria), (3) alterations inintestinal oxalate-degrading microorganisms, and (4) genetic variationsof intestinal oxalate transporters. Furthermore, hyperoxaluria may alsooccur following renal transplantation because of rapid clearance ofaccumulated oxalate.

Accordingly, there is a need in the art for effective methods fortreating hyperoxaluria.

SUMMARY OF THE INVENTION

The present invention provides polynucleotide agents, e.g., antisensepolynucleotide agents, and compositions comprising such agents whichtarget nucleic acids encoding hydroxyacid oxidase (HAO1) and interferewith the normal function of the targeted nucleic acid. The HAO1 nucleicacid may be within a cell, e.g., a cell within a subject, such as ahuman. The present invention also provides methods and combinationtherapies for treating a subject having a disorder that would benefitfrom inhibiting or reducing the expression of a HAO1 mRNA, e.g., a HAO1associated disease, such as primary or secondary hyperoxaluria, usingthe polynucleotide agents and compositions of the invention.

Accordingly, in one aspect, the present invention providespolynucleotide agents for inhibiting expression of a hydroxyacid oxidase(HAO1) gene. The agents include about 4 to about 50 contiguousnucleotides, wherein at least one of the contiguous nucleotides is amodified nucleotide, and wherein the nucleotide sequence of the agent isabout 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%,or about 100% complementary over its entire length to the equivalentregion of the nucleotide sequence of any one of SEQ ID NOs:1-4.

In another aspect, the present invention provides polynucleotide agentsfor inhibiting expression of a hydroxyacid oxidase (HAO1) gene. Theagents include at least 8 contiguous nucleotides differing by no morethan 3 nucleotides from any one of the target nucleotide sequences ofSEQ ID NO:1 provided in Tables 3 and 4 and are about 8 to about 50nucleotides in length.

In some embodiments, substantially all of the nucleotides of thepolynucleotide agents of the invention are modified nucleotides. Inother embodiments, all of the nucleotides of the polynucleotide agentare modified nucleotides.

The polynucleotide agent may be 10 to 40 nucleotides in length; 10 to 30nucleotides in length; 18 to 30 nucleotides in length; 10 to 24nucleotides in length; 18 to 24 nucleotides in length; or 20 nucleotidesin length.

In one embodiment, the modified nucleotide comprises a modified sugarmoiety selected from the group consisting of a 2′-O-methoxyethylmodified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkylmodified sugar moiety, and a bicyclic sugar moiety.

In one embodiment, the bicyclic sugar moiety has a (—CRH—)n groupforming a bridge between the 2′ oxygen and the 4′ carbon atoms of thesugar ring, wherein n is 1 or 2 and wherein R is H, CH₃ or CH₃OCH₃.

In a further embodiment, n is 1 and R is CH₃.

In another embodiment, the modified nucleotide is a 5-methylcytosine.

In one embodiment, the modified nucleotide comprises a modifiedinternucleoside linkage, such as a phosphorothioate internucleosidelinkage.

In one embodiment, the polynucleotide agent of the invention comprisesone 2′-deoxynucleotide. In another embodiment, the polynucleotide agentof the invention comprises one 2′-deoxynucleotide flanked on each sideby at least one nucleotide having a modified sugar moiety.

In one embodiment, the polynucleotide agent of the invention comprises aplurality, e.g., more than 1, e.g., 2, 3, 4, 5, 6, or 7,2′-deoxynucleotides. In one embodiment, the polynucleotide agent of theinvention comprises a plurality, e.g., more than 1, e.g., 2, 3, 4, 5, 6,or 7, 2′-deoxynucleotides flanked on each side by at least onenucleotide having a modified sugar moiety.

In one embodiment, the agent is a gapmer comprising a gap segmentcomprised of linked 2′-deoxynucleotides positioned between a 5′ and a 3′wing segment.

In one embodiment, the modified sugar moiety is selected from the groupconsisting of a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, and abicyclic sugar moiety.

In one embodiment, the 5′-wing segment is 1 to 6 nucleotides in length,e.g., 2, 3, 4, or 5 nucleotides in length.

In one embodiment, the 3′-wing segment is 1 to 6 nucleotides in length,e.g., 2, 3, 4, or 5 nucleotides in length.

In one embodiment, the gap segment is 5 to 14 nucleotides in length,e.g., 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. In oneembodiment, the gap segment is 10 nucleotides in length.

In one aspect, the present invention provides polynucleotide agents,e.g., antisense polynucleotide agents, for inhibiting expression of ahydroxyacid oxidase (HAO1) gene. The agents include a gap segmentconsisting of linked deoxynucleotides; a 5′-wing segment consisting oflinked nucleotides; a 3′-wing segment consisting of linked nucleotides;wherein the gap segment is positioned between the 5′-wing segment andthe 3′-wing segment and wherein each nucleotide of each wing segmentcomprises a modified sugar.

In one embodiment, the gap segment is ten 2′-deoxynucleotides in lengthand each of the wing segments is five nucleotides in length.

In another embodiment, the gap segment is ten 2′-deoxynucleotides inlength and each of the wing segments is four nucleotides in length.

In yet another embodiment, the gap segment is ten 2′-deoxynucleotides inlength and each of the wing segments is three nucleotides in length.

In another embodiment, the gap segment is ten 2′-deoxynucleotides inlength and each of the wing segments is two nucleotides in length.

In one embodiment, the modified sugar moiety is selected from the groupconsisting of a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, and abicyclic sugar moiety.

In some embodiments, the agents of the invention further comprise aligand.

In one embodiment, the agent is conjugated to the ligand at the3′-terminus.

In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc)derivative. In certain embodiments, the ligand is one or more GalNAcderivatives attached through a monovalent, a bivalent, or a trivalentbranched linker. The ligand may be conjugated to the 3′ end of the sensestrand of the polynucleotide agent, the 5′ end of the sense strand ofthe polynucleotide agent, the 3′ end of the antisense strand of thepolynucleotide agent, or the 5′ end of the antisense strand of thepolynucleotide agent.

In some embodiments, the polynucleotide agents of the invention comprisea plurality, e.g., 2, 3, 4, 5, or 6, of GalNAc, each independentlyattached to a plurality of nucleotides of the polynucleotide agentthrough a plurality of monovalent linkers.

In one embodiment, the ligand is

In one aspect, the present invention provides pharmaceuticalcompositions for inhibiting expression of a hydroxyacid oxidase (HAO1)gene comprising a polynucleotide agent of the invention.

In one embodiment, the agent is present in an unbuffered solution, suchas saline or water.

In another embodiment, the agent is is present in a buffer solution,such as a buffer comprising acetate, citrate, prolamine, carbonate, orphosphate or any combination thereof.

In one embodiment, the buffer solution is phosphate buffered saline(PBS).

In another aspect, the present invention provides pharmaceuticalcomposition comprising a polynucleotide agent of the invention and alipid formulation, such as a lipid formulation comprising an LNP or aMC3.

In one aspect, the present invention provides methods of inhibitingexpression of a hydroxyacid oxidase (HAO1) gene in a cell. The methodsinclude contacting the cell with a polynucleotide agent of the inventionor a pharmaceutical composition of the invention; and maintaining thecell for a time sufficient to obtain antisense inhibition of a HAO1gene, thereby inhibiting expression of the HAO1 gene in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human.

In one embodiment, the HAO1 expression is inhibited by at least about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 98% or about 100%.

In another aspect, the present invention provides methods of treating asubject having a disease or disorder that would benefit from reductionin hydroxyacid oxidase (HAO1) expression. The methods includeadministering to the subject a therapeutically effective amount of apolynucleotide agent of the invention or a pharmaceutical composition ofthe invention, thereby treating the subject.

In yet another aspect, the present invention provides methods ofpreventing at least one symptom in a subject having a disease ordisorder that would benefit from reduction in hydroxyacid oxidase (HAO1)expression. The methods include administering to the subject aprophylactically effective amount of a polynucleotide agent of theinvention or a pharmaceutical composition of the invention, therebypreventing at least one symptom in the subject having a disorder thatwould benefit from reduction in HAO1 expression.

In one embodiment, the administration of the polynucleotide agent to thesubject causes a decrease oxalate levels and/or a decrease in HAO1protein levels in the subject.

In one embodiment, the disorder is a HAO1-associated disease.

In certain embodiments, the HAO1-associated disease is selected from thegroup consisting of primary hyperoxaluria and secondary hyperoxaluria.

In one embodiment, the HAO1-associated disease is primary hyperoxaluria.In further embodiments, the primary hyperoxaluria is selected from thegroup consisting of primary hyperoxaluria type 1 (PH1), primaryhyperoxaluria type 2 (PH2) and primary hyperoxaluria type 3 (PH3).

In other embodiment, the HAO1-associated disease is secondaryhyperoxaluria. In further aspects, the secondary hyperoxaluria isenteric hyperoxaluria or hyperoxaluria resulting from high oxalate diet,deficiencies in intestinal oxalate degrading microorganisms, geneticvariations in intestinal oxalate transporters or renal transplantation.

In certain embodiments the subject is human.

In certain embodiments, the methods further comprise administering tothe subject an additional therapeutic agent. In some embodiments, theadditional therapeutic agent is selected from the group consisting of asuch as vitamin B6 (pyridoxine) and/or potassium citrate, or acombination thereof.

In certain embodiments, the agent is administered at a dose of about0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

In certain embodiments, the agent is administered at a dose of about 10mg/kg to about 30 mg/kg.

In certain embodiments, the agent is administered to the subject once aweek.

In alternative embodiments, the agent is administered to the subjecttwice a week.

In yet other embodiments, the agent is administered to the subject twicea month.

In certain embodiments, the agent is administered to the subjectsubcutaneously.

In certain embodiments, the methods of the invention further includemeasuring HAO1 levels in the subject and/or oxalate levels in thesubject's urine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the endogenous pathway for oxalate synthesis(from Robijn, et al. (2011) Kidney International 80:1146-1158).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polynucleotide agents, e.g., antisensepolynucleotide agents, and compositions comprising such agents whichtarget nucleic acids encoding hydroxyacid oxidase (HAO1) (e.g., mRNAencoding HAO1 as provided in, for example, any one of SEQ ID NOs:1-4).The polynucleotide agents, e.g., antisense polynucleotide agents, bindto nucleic acids encoding HAO1 via, e.g., Watson-Crick base pairing, andinterfere with the normal function of the targeted nucleic acid.

The polynucleotide agents of the invention include a nucleotide sequencewhich is about 4 to about 50 nucleotides or less in length and which isabout 80% complementary to at least part of an mRNA transcript of anHAO1 gene. The use of these polynucleotide agents enables the targetedinhibition of RNA expression and/or activity of an HAO1 gene in mammals.

The present inventors have demonstrated that polynucleotide agents,e.g., antisense polynucleotide agents, targeting HAO1 can mediateantisense inhibition in vitro resulting in significant inhibition ofexpression of a HAO1 gene. Thus, methods and compositions includingthese polynucleotide agents are useful for treating a subject who wouldbenefit by a reduction in the levels and/or activity of an HAO1 protein,such as a subject having a HAO1-associated disease, such as primary orsecondary hyperoxaluria.

The present invention also provides methods and combination therapiesfor treating a subject having a disorder that would benefit frominhibiting or reducing the expression of an HAO1 gene, e.g., anHAO1-associated disease, such as primary or secondary hyperoxaluriausing the polynucleotide agents and compositions of the invention.

The present invention also provides methods for preventing at least onesymptom, e.g., oxalate accumulation, in a subject having a disorder thatwould benefit from inhibiting or reducing the expression of an HAO1gene, e.g., primary or secondary hyperoxaluria. The present inventionfurther provides compositions comprising polynucleotide agents whicheffect inhibition of an HAO1 gene. The HAO1 gene may be within a cell,e.g., a cell within a subject, such as a human.

The combination therapies of the present invention include administeringto a subject having a HAO1-associated disease, a polynucleotide agent ofthe invention, e.g., an antisense polynucleotide agent, and anadditional therapeutic, such as vitamin B6 (pyridoxine) and/or potassiumcitrate or a combination of any of the foregoing. The combinationtherapies of the invention reduce HAO1 levels in the subject (e.g., byabout 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or about 99%) by targeting HAO1 mRNA with a polynucleotide agent ofthe invention and, accordingly, allow the therapeutically (orprophylactically) effective amount of the additional therapeuticagent(s) required to treat the subject to be reduced, thereby decreasingthe costs of treatment and permitting easier and more convenient ways ofadministering certain agents, or decreasing side effects of certainagents.

The following detailed description discloses how to make and usepolynucleotide agents to inhibit the mRNA and/or protein expression ofan HAO1 gene, as well as compositions, uses, and methods for treatingsubjects having diseases and disorders that would benefit frominhibition and/or reduction of the expression of this gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges oftolerances in the art.

As used herein, “hydroxyacid oxidase,” used interchangeably with theterms “HAO1”, “glycolate oxidase” and “GO”, refers to the well-knowngene and polypeptide, also known in the art as as glycolate oxidase and(S)-2-hydroxy-acid oxidase.

The term “HAO1” includes human HAO1, the amino acid and complete codingsequence of which may be found in for example, GenBank Accession No. GI:11184232 (NM_017545.2; SEQ ID NO:1); Macaca fascicularis HAO1, the aminoacid and complete coding sequence of which may be found in for example,GenBank Accession No. GI: 544464345 (XM_005568381.1: SEQ ID NO:2); mouse(Mus musculus) HAO1, the amino acid and complete coding sequence ofwhich may be found in for example, GenBank Accession No. GI: 133893166(NM_010403.2; SEQ ID NO:3); and rat HAO1 (Rattus norvegicus) HAO1 theamino acid and complete coding sequence of which may be found in forexample, for example GenBank Accession No. GI: 166157785 (NM_001107780;SEQ ID NO:4).

Additional examples of HAO1 mRNA sequences are readily available usingpublicly available databases, e.g., GenBank, UniProt, OMIM, and theMacaca genome project web site.

The term“HAO1,” as used herein, also refers to naturally occurring DNAsequence variations of the HAO1 gene, such as a single nucleotidepolymorphism (SNP) in the HAO1 gene. Exemplary SNPs may be found in thedbSNP database available at ncbi.nlm.nih.gov/projects/SNP/.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a HAO1 gene, including mRNA that is a product of RNA processing of aprimary transcription product.

As used herein, “target nucleic acid” refers to a nucleic acid moleculeto which a polynucleotide agent, e.g., an antisense polynucleotideagent, of the invention specifically hybridizes.

The terms “polynucleotide agent” “compound”, and “agent” as usedinterchangeably herein, refer to an agent comprising a single-strandedoligonucleotide that contains RNA as that term is defined herein, andwhich targets nucleic acid molecules encoding HAO1 (e.g., mRNA encodingHAO1 as provided in, for example, any one of SEQ ID NOs:1-4). Thepolynucleotide agents, e.g., antisense polynucleotide agents,specifically bind to the target nucleic acid molecules via hydrogenbonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding) and interfere with the normal function of the targeted nucleicacid (e.g., by an antisense mechanism of action). This interference withor modulation of the function of a target nucleic acid by thepolynucleotide agents of the present invention is referred to as“antisense inhibition.”

The functions of the target nucleic acid molecule to be interfered withmay include functions such as, for example, translocation of the RNA tothe site of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA.

In some embodiments, antisense inhibition refers to “inhibiting theexpression” of target nucleic acid levels and/or target protein levelsin a cell, e.g., a cell within a subject, such as a mammalian subject,in the presence of the polynucleotide agent complementary to a targetnucleic acid as compared to target nucleic acid levels and/or targetprotein levels in the absence of the polynucleotide agent. For example,the polynucleotide agents of the invention can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355.

As used herein, the term “specifically hybridizes” refers to apolynucleotide agent having a sufficient degree of complementaritybetween the polynucleotide agent and a target nucleic acid to induce adesired effect, while exhibiting minimal or no effects on non-targetnucleic acids under conditions in which specific binding is desired,e.g., under physiological conditions in the case of in vivo assays andtherapeutic treatments.

A target sequence may be from about 4-50 nucleotides in length, e.g.,8-45, 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30,11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35,13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45,15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25,16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35,18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous nucleotides of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an HAO1 gene. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” are used herein with respect to the base matching betweena polynucleotide agent and a target sequence. The term“complementarity”refers to the capacity for pairing between nucleobases of a firstnucleic acid and a second nucleic acid.

As used herein, a polynucleotide agent that is “substantiallycomplementary to at least part of” a messenger RNA (mRNA) refers to apolynucleotide agent that is substantially complementary to a contiguousportion of the mRNA of interest (e.g., an mRNA encoding HAO1). Forexample, a polynucleotide is complementary to at least a part of an HAO1mRNA if the sequence is substantially complementary to a non-interruptedportion of an mRNA encoding HAO1.

As used herein, the term “region of complementarity” refers to theregion of the polynucletiode agent that is substantially complementaryto a sequence, for example a target sequence, e.g., an HAO1 nucleotidesequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the polynucleotide.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of apolynucleotide comprising the first nucleotide sequence to hybridize andform a duplex structure under certain conditions with the secondnucleotide sequence, as will be understood by the skilled person. Suchconditions can, for example, be stringent conditions, where stringentconditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. for 12-16 hours followed by washing (see, e.g., “MolecularCloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring HarborLaboratory Press). Other conditions, such as physiologically relevantconditions as can be encountered inside an organism, can apply. Theskilled person will be able to determine the set of conditions mostappropriate for a test of complementarity of two sequences in accordancewith the ultimate application of the nucleotides.

Complementary sequences include those nucleotide sequences of apolynucleotide agent of the invention that base-pair to a secondnucleotide sequence over the entire length of one or both nucleotidesequences. Such sequences can be referred to as “fully complementary”with respect to each other herein. However, where a first sequence isreferred to as “substantially complementary” with respect to a secondsequence herein, the two sequences can be fully complementary, or theycan form one or more, but generally not more than 5, 4, 3 or 2mismatched base pairs upon hybridization for a duplex up to 30 basepairs, while retaining the ability to hybridize under the conditionsmost relevant to their ultimate application, e.g., antisense inhibitionof target gene expression.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the terms“deoxyribonucleotide”, “ribonucleotide” and “nucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety (see, e.g., Table 2). The skilled person is wellaware that guanine, cytosine, adenine, and uracil can be replaced byother moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base can base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine can be replaced in the nucleotide sequences of theagents featured in the invention by a nucleotide containing, forexample, inosine. In another example, adenine and cytosine anywhere inthe oligonucleotide can be replaced with guanine and uracil,respectively to form G-U Wobble base pairing with the target mRNA.Sequences containing such replacement moieties are suitable for thecompositions and methods featured in the invention.

A “nucleoside” is a base-sugar combination. The “nucleobase” (also knownas “base”) portion of the nucleoside is normally a heterocyclic basemoiety. “Nucleotides” are nucleosides that further include a phosphategroup covalently linked to the sugar portion of the nucleoside. Forthose nucleosides that include a pentofuranosyl sugar, the phosphategroup can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.“Polynucleotides,” also referred to as “oligonucleotides,” are formedthrough the covalent linkage of adjacent nucleosides to one another, toform a linear polymeric oligonucleotide. Within the polynucleotidestructure, the phosphate groups are commonly referred to as forming theinternucleoside linkages of the polynucleotide.

In general, the majority of nucleotides of the polynucleotide agents areribonucleotides, but as described in detail herein, the agents may alsoinclude one or more non-ribonucleotides, e.g., a deoxyribonucleotide. Inaddition, as used in this specification, a “polynucleotide agent” mayinclude nucleotides (e.g., ribonucleotides or deoxyribonucleotides) withchemical modifications; a polynucleotide agent may include substantialmodifications at multiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, and/or modified nucleobase. Thus, the termmodified nucleotide encompasses substitutions, additions or removal of,e.g., a functional group or atom, to internucleoside linkages, sugarmoieties, or nucleobases. The modifications suitable for use in thepolynucleotide agents of the invention include all types ofmodifications disclosed herein or known in the art. Any suchmodifications, as used in nucleotides, are encompassed by“polynucleotide agent” for the purposes of this specification andclaims.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in HAO1 expression; a human at risk for adisease, disorder or condition that would benefit from reduction in HAO1expression; a human having a disease, disorder or condition that wouldbenefit from reduction in HAO1 expression; and/or human being treatedfor a disease, disorder or condition that would benefit from reductionin HAO1 expression as described herein.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with unwanted HAO1expression, e.g., hyperoxaluria, nephrocalcinosis and/ornephrolithiasis. “Treatment” can also mean prolonging survival ascompared to expected survival in the absence of treatment.

The term “lower” in the context of the level of HAO1 in a subject or adisease marker or symptom refers to a statistically significant decreasein such level. The decrease can be, for example, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more. In certain embodiments, a decrease is atleast 20%. “Lower” in the context of the level of HAO1 in a subject ispreferably down to a level accepted as within the range of normal for anindividual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an HAO1 gene, refers to a reduction in thelikelihood that a subject will develop a symptom associated with such adisease, disorder, or condition, e.g., a symptom associated with adisease or disorder that is caused by, or associated with HAO1expression, e.g., hyperoxaluria, nephrocalcinosis and/ornephrolithiasis. The likelihood of developing hyperoxaluria is reduced,for example, when an individual having one or more risk factors forhyperoxaluria either fails to develop hyperoxaluria or developshyperoxaluria with less severity relative to a population having thesame risk factors and not receiving treatment as described herein. Thefailure to develop a disease, disorder or condition, or the reduction inthe development of a symptom associated with such a disease, disorder orcondition (e.g., by at least about 10% on a clinically accepted scalefor that disease or disorder), or the exhibition of delayed symptomsdelayed (e.g., by days, weeks, months or years) is considered effectiveprevention.

As used herein, the term “hydroxyacid oxidase-associated disease” or“HAO1-associated disease,” is a disease or disorder that is caused by,or associated with the activity of HAO1 protein and the symptoms orprogression of which respond to HAO1 inactivation. The term“HAO1-associated disease” includes a disease, disorder or condition thatwould benefit from reduction in HAO1 expression. Such diseases aretypically associated with elevated levels of oxalate, or hyperoxaluria.Non-limiting examples of such diseases include primary hyperoxaluria andsecondary hyperoxaluria.

Primary Hyperoxaluria

The primary hyperoxalurias, which may be of type 1 (PH1), type 2 (PH2)and type 3, are relatively rare autosomal recessive disorders ofglyoxylate metabolism, which are characterized by markedly increasedendogenous oxalate levels. All three types are characterized by theinability to remove glyoxylate, as is shown in FIG. 1. PH1, accountingfor the majority of cases (70-80%), results from the absence ordeficiency of the peroxisomal liver enzyme AGT, the activity of whichdepends on pyridoxal phosphate. As AGT catalyzes the transamination ofglyoxylate to glycine, its deficiency in PH1 allows glyoxylate to bereduced to glycolate and to be oxidized to oxalate. PH2 results from thedeficiency of the cytosolic liver enzyme glyoxylatereductase/hydroxypyruvate reductase (GRHPR). Severe hyperoxaluria is theclinical hallmark of PH1 and PH2, with reported urine oxalate levelsranging between 88 and 352 mg per 24 h (1-4 mmol per 24 h) for PH1 and88 and 176 mg per 24 h (1-2 mmol per 24 h) for PH2.

In a third form of hyperoxaluria, PH3, patients present with normal AGTand GRHPR enzyme activities. Without wishing to be bound by a specifictheory, it is believed that mutations in DHDPSL are responsible for PH3.It is assumed that DHDPSL encodes a 4-hydroxy-2-oxoglutarate aldolasewhich catalyzes the final step in the metabolism of hydroxyproline (seeFIG. 1).

Secondary Hyperoxaluria

Secondary hyperoxaluria may be caused by a variety of conditions, whichinclude fat malabsorption (enteric hyperoxaluria) or hyperoxaluriaresulting from high oxalate diet, deficiencies in intestinal oxalatedegrading microorganisms, genetic variations in intestinal oxalatetransporters or renal transplantation.

High-oxalate diet. Dietary oxalate is an important determining factor inurinary oxalate excretion. Average daily oxalate intake of the westernpopulation may range from about 44 and about 351 mg/day (0.5-4.0mmol/day), and may even exceed 1000 mg/day (11.4 mmol/day) whenoxalate-rich foods, such as spinach or rhubarb, are consumed. However,the fraction of dietary oxalate that will effectively be absorbed by theintestine is highly influenced by the amount of oxalate-binding cations,such as calcium and magnesium, in the gut. Concomitant ingestion ofcalcium (or magnesium) with oxalate can reduce oxaluria by forminginsoluble oxalate complexes in the gut, thereby decreasing intestinaloxalate absorption. Other parameters that can influence oxalateabsorption include, but are not limited to, oxalate bioavailability,amount of oxalate precursors, inherited oxalate absorption capacity,gastric emptying, intestinal transit time, and the presence ofoxalate-degrading microorganisms. Studies showed that an oxalate loadresults in transiently increased plasma and urine oxalate levels peaking2 to 4 h post load, implying that an oxalate-rich meal is able to inducetemporary states of hyperoxaluria.

Fat Malabsorption (Enteric Hyperoxaluria).

Hyperoxaluria due to fat malabsorption refers to a condition in whichintestinal oxalate absorption is increased as a result of two differentmechanisms: (1) both dihydroxy bile acids and fatty acids increase thepermeability of the intestinal mucosa to oxalate and (2) complexation offatty acids with luminal calcium increases the amount of soluble oxalatethat is available for absorption as insoluble CaOx complexes are nolonger formed. Hyperoxaluria due to fat malabsorption is typically seenin patients suffering from inflammatory bowel disorders, after bariatricsurgery (potentially leading to kidney failure or after the use ofgastrointestinal lipase inhibitors.

Deficiencies in Intestinal Oxalate-Degrading Microorganisms.

One of the best-known oxalate-degrading organisms is Oxalobacterformigenes, a Gram-negative anaerobic bacterium that is found in thecolon of humans and other vertebrates and that relies exclusively on theconversion of oxalate to formate as its energy source. Oxalate entersthe bacterium through an oxalate-formate antiporter on the cellmembrane, where it is metabolized to formate and CO₂, resulting in aproton gradient used to drive ATP synthesis. Without wishing to be boundby a specific theory, it is believed that colonization of the gut withO. formigenes reduces intestinal oxalate absorption, and hence decreasesurinary oxalate excretion.

Genetic Variations of Intestinal Oxalate Transporters.

Without wishing to be bound by a specific theory, it is believed, basedon the recent studies, that variations in intestinal oxalatetransporters may be associated with reduced intestinal oxalate secretionand increased prevalence or severity of nephrocalcinosis and/ornephrolithiasis. Recent studies have shown that deletion of the slc26a6oxalate transporter gene in mice, a species virtually insensitive tolithogenic agents, results in hyperoxalemia, hyperoxaluria, and CaOxurolithiasis due to a defect in intestinal oxalate secretion.

Hyperoxaluria Following Renal Transplantation.

In patients with impared renal function and renal failure, glomerularfiltration rate declines and oxalate clearance becomes compromised,resulting in elevated plasma oxalate levels that may be up to 10 timesabove normal in predialysis patients. As CaOx supersaturation mayalready occur at plasma oxalate levels of 30 μmol/l, uremic plasma isoften supersaturated, potentially leading to systemic oxalosis.Following renal transplantation or combined liver/kidneytransplantation, the accumulated oxalate is rapidly released from thebody, resulting in transient hyperoxaluria and risk of CaOxprecipitation within the allograft tissue, especially in the presence ofallograft dysfunction.

II. Polynucleotide Agents of the Invention

The present invention provides polynucleotide agents, e.g., antisensepolynucleotide agents, and compositions comprising such agents, whichtarget an HAO1 gene and inhibit the expression of the HAO1 gene. In oneembodiment, the polynucleotide agents inhibit the expression of an HAO1gene in a cell, such as a cell within a subject, e.g., a mammal, such asa human having an HAO1-associated disease, e.g., primary or secondaryhyperoxaluria.

The polynucleotide agents of the invention include a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of an HAO1 gene. The region of complementaritymay be about 50 nucleotides or less in length (e.g., about 50, 49, 48,47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, or 4 nucleotides or less in length). Upon contactwith a cell expressing the HAO1 gene, the polynucleotide agent inhibitsthe expression of the HAO1 gene (e.g., a human, a primate, anon-primate, or a bird HAO1 gene) by at least about 10% as assayed by,for example, a PCR or branched DNA (bDNA)-based method, or by aprotein-based method, such as by immunofluorescence analysis, using, forexample, western Blotting or flow cytometric techniques.

The region of complementarity between a polynucleotide agent and atarget sequence may be substantially complementary (e.g., there is asufficient degree of complementarity between the polynucleotide agentand a target nucleic acid to so that they specifically hybridize andinduce a desired effect), but is generally fully complementary to thetarget sequence. The target sequence can be derived from the sequence ofan mRNA formed during the expression of a HAO1 gene.

Accordingly, in one aspect, a polynucleotide agent of the invention,e.g., an antisense polynucleotide agent, specifically hybridizes to atarget nucleic acid molecule, such as the mRNA encoding HAO1, andcomprises a contiguous nucleotide sequence which corresponds to thereverse complement of a nucleotide sequence of any one of SEQ IDNOs:1-4, or a fragment of any one of SEQ ID NOs:1-4.

In some embodiments, the polynucleotide agents of the invention may besubstantially complementary to the target sequence. For example, apolynucleotide agent that is substantially complementary to the targetsequence may include a contiguous nucleotide sequence comprising no morethan 5 mismatches (e.g., no more than 1, no more than 2, no more than 3,no more than 4, or no more than 5 mismatches) when hybridizing to atarget sequence, such as to the corresponding region of a nucleic acidwhich encodes a mammalian HAO1 mRNA. In some embodiments, the contiguousnucleotide sequence comprises no more than a single mismatch whenhybridizing to the target sequence, such as the corresponding region ofa nucleic acid which encodes a mammalian HAO1 mRNA.

In some embodiments, the polynucleotide agents of the invention that aresubstantially complementary to the target sequence comprise a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of SEQ ID NOs:1-4, or a fragment of any one of SEQ ID NOs:1-4, suchas about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary.

In some embodiments, a polynucleotide agent comprises a contiguousnucleotide sequence which is fully complementary over its entire lengthto the equivalent region of the nucleotide sequence of any one of SEQ IDNOs:1-4 (or a fragment of any one of SEQ ID NOs:1-4).

A polynucleotide agent may comprise a contiguous nucleotide sequence ofabout 4 to about 50 nucleotides in length, e.g., 8-49, 8-48, 8-47, 8-46,8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34,8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22,8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10,8-9, 10-49, 10-48, 10-47, 10-46, 10-45, 10-44, 10-43, 10-42, 10-41,10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32, 10-31,10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21,10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11,11-49, 11-48, 11-47, 11-46, 11-45, 11-44, 11-43, 11-42, 11-41, 11-40,11-39, 11-38, 11-37, 11-36, 11-35, 11-34, 11-33, 11-32, 11-31, 11-30,11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21, 11-20,11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-49, 12-48,12-47, 12-46, 12-45, 12-44, 12-43, 12-42, 12-41, 12-40, 12-39, 12-38,12-37, 12-36, 12-35, 12-34, 12-33, 12-32, 12-31, 12-30, 12-29, 12-28,12-27, 12-26, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 12-19, 12-18,12-17, 12-16, 12-15, 12-14, 12-13, 13-49, 13-48, 13-47, 13-46, 13-45,13-44, 13-43, 13-42, 13-41, 13-40, 13-39, 13-38, 13-37, 13-36, 13-35,13-34, 13-33, 13-32, 13-31, 13-30, 13-29, 13-28, 13-27, 13-26, 13-25,13-24, 13-23, 13-22, 13-21, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15,13-14, 14-49, 14-48, 14-47, 14-46, 14-45, 14-44, 14-43, 14-42, 14-41,14-40, 14-39, 14-38, 14-37, 14-36, 14-35, 14-34, 14-33, 14-32, 14-31,14-30, 14-29, 14-28, 14-27, 14-26, 14-25, 14-24, 14-23, 14-22, 14-21,14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-49, 15-48, 15-47, 15-46,15-45, 15-44, 15-43, 15-42, 15-41, 15-40, 15-39, 15-38, 15-37, 15-36,15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 15-16,16-49, 16-48, 16-47, 16-46, 16-45, 16-44, 16-43, 16-42, 16-41, 16-40,16-39, 16-38, 16-37, 16-36, 16-35, 16-34, 16-33, 16-32, 16-31, 16-30,16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 16-23, 16-22, 16-21, 16-20,16-19, 16-18, 16-17, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43,17-42, 17-41, 17-40, 17-39, 17-38, 17-37, 17-36, 17-35, 17-34, 17-33,17-32, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-23,17-22, 17-21, 17-20, 17-19, 17-18, 18-49, 18-48, 18-47, 18-46, 18-45,18-44, 18-43, 18-42, 18-41, 18-40, 18-39, 18-38, 18-37, 18-36, 18-35,18-34, 18-33, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,18-24, 18-23, 18-22, 18-21, 18-20, 19-49, 19-48, 19-47, 19-46, 19-45,19-44, 19-43, 19-42, 19-41, 19-40, 19-39, 19-38, 19-37, 19-36, 19-35,19-34, 19-33, 19-32, 19-31, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25,19-24, 19-23, 19-22, 19-21, 19-20, 20-49, 20-48, 20-47, 20-46, 20-45,20-44, 20-43, 20-42, 20-41, 20-40, 20-39, 20-38, 20-37, 20-36, 20-35,20-34, 20-33, 20-32, 20-31, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25,20-24, 20-23, 20-22, 20-21, 21-49, 21-48, 21-47, 21-46, 21-45, 21-44,21-43, 21-42, 21-41, 21-40, 21-39, 21-38, 21-37, 21-36, 21-35, 21-34,21-33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, 21-22, 22-49, 22-48, 22-47, 22-46, 22-45, 22-44, 22-43, 22-42,22-41, 22-40, 22-39, 22-38, 22-37, 22-36, 22-35, 22-34, 22-33, 22-32,22-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 23-49,23-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40, 23-39,23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30, 23-29,23-28, 23-27, 23-26, 23-25, 23-24, 24-49, 24-48, 24-47, 24-46, 24-45,24-44, 24-43, 24-42, 24-41, 24-40, 24-39, 24-38, 24-37, 24-36, 24-35,24-34, 24-33, 24-32, 24-31, 24-30, 24-29, 24-28, 24-27, 24-26, 24-25,25-49, 25-48, 25-47, 25-46, 25-45, 25-44, 25-43, 25-42, 25-41, 25-40,25-39, 25-38, 25-37, 25-36, 25-35, 25-34, 25-33, 25-32, 25-31, 25-30,25-29, 25-28, 25-27, 25-26, 26-49, 26-48, 26-47, 26-46, 26-45, 26-44,26-43, 26-42, 26-41, 26-40, 26-39, 26-38, 26-37, 26-36, 26-35, 26-34,26-33, 26-32, 26-31, 26-30, 26-29, 26-28, 26-27, 27-49, 27-48, 27-47,27-46, 27-45, 27-44, 27-43, 27-42, 27-41, 27-40, 27-39, 27-38, 27-37,27-36, 27-35, 27-34, 27-33, 27-32, 27-31, 27-30, 27-29, 27-28, 28-49,28-48, 28-47, 28-46, 28-45, 28-44, 28-43, 28-42, 28-41, 28-40, 28-39,28-38, 28-37, 28-36, 28-35, 28-34, 28-33, 28-32, 28-31, 28-30, 28-29,29-49, 29-48, 29-47, 29-46, 29-45, 29-44, 29-43, 29-42, 29-41, 29-40,29-39, 29-38, 29-37, 29-36, 29-35, 29-34, 29-33, 29-32, 29-31, 29-30,30-49, 30-48, 30-47, 30-46, 30-45, 30-44, 30-43, 30-42, 30-41, 30-40,30-39, 30-38, 30-37, 30-36, 30-35, 30-34, 30-33, 30-32, or 30-31nucleotides in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length.

In some embodiments, a polynucleotide agent may comprise a contiguousnucleotide sequence of no more than 22 nucleotides, such as no more than21 nucleotides, 20 nucleotides, 19 nucleotides, or no more than 18nucleotides. In some embodiments the polynucleotide agenst of theinvention comprises less than 20 nucleotides. In other embodiments, thepolynucleotide agents of the invention comprise 20 nucleotides.

In certain aspects, a polynucleotide agent of the invention includes asequence selected from the group of sequences provided in Tables 3 and4. It will be understood that, although some of the sequences in Table 3are described as modified and/or conjugated sequences, a polynucleotideagent of the invention, may also comprise any one of the sequences setforth in Table 3 that is un-modified, un-conjugated, and/or modifiedand/or conjugated differently than described therein.

By virtue of the nature of the nucleotide sequences provided in Tables 3and 4, polynucleotide agents of the invention may include one of thesequences of Tables 3 and 4 minus only a few nucleotides on one or bothends and yet remain similarly effective as compared to thepolynucleotide agents described above. Hence, polynucleotide agentshaving a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more contiguous nucleotides derived from one ofthe sequences of Tables 3 and 4 and differing in their ability toinhibit the expression of an HAO1 gene by not more than about 5, 10, 15,20, 25, or 30% inhibition from an polynucleotide agent comprising thefull sequence, are contemplated to be within the scope of the presentinvention.

In addition, the polynucleotide agents provided in Tables 3 and 4identify a region(s) in an HAO1 transcript that is susceptible toantisense inhibition (e.g., the regions in SEQ ID NO: 1 which thepolynucleotide agents may target). As such, the present inventionfurther features polynucleotide agents that target within one of thesesites. As used herein, a polynucleotide agent is said to target within aparticular site of an RNA transcript if the polynucleotide agentpromotes antisense inhibition of the target at that site. Such apolynucleotide agent will generally include at least about 15 contiguousnucleotides from one of the sequences provided in Tables 3 and 4 coupledto additional nucleotide sequences taken from the region contiguous tothe selected sequence in an HAO1 gene.

While a target sequence is generally about 4-50 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing antisense inhibition of any given target RNA.Various software packages and the guidelines set out herein provideguidance for the identification of optimal target sequences for anygiven gene target, but an empirical approach can also be taken in whicha “window” or “mask” of a given size (as a non-limiting example, 20nucleotides) is literally or figuratively (including, e.g., in silico)placed on the target RNA sequence to identify sequences in the sizerange that can serve as target sequences. By moving the sequence“window” progressively one nucleotide upstream or downstream of aninitial target sequence location, the next potential target sequence canbe identified, until the complete set of possible sequences isidentified for any given target size selected. This process, coupledwith systematic synthesis and testing of the identified sequences (usingassays as described herein or as known in the art) to identify thosesequences that perform optimally can identify those RNA sequences that,when targeted with a polynucleotide agent, mediate the best inhibitionof target gene expression. Thus, while the sequences identified, forexample, in Tables 3 and 4, represent effective target sequences, it iscontemplated that further optimization of antisense inhibitionefficiency can be achieved by progressively “walking the window” onenucleotide upstream or downstream of the given sequences to identifysequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 3 and 4, further optimization could be achieved by systematicallyeither adding or removing nucleotides to generate longer or shortersequences and testing those sequences generated by walking a window ofthe longer or shorter size up or down the target RNA from that point.Again, coupling this approach to generating new candidate targets withtesting for effectiveness of polynucleotide agents based on those targetsequences in an inhibition assay as known in the art and/or as describedherein can lead to further improvements in the efficiency of inhibition.Further still, such optimized sequences can be adjusted by, e.g., theintroduction of modified nucleotides as described herein or as known inthe art, addition or changes in length, or other modifications as knownin the art and/or discussed herein to further optimize the molecule(e.g., increasing serum stability or circulating half-life, increasingthermal stability, enhancing transmembrane delivery, targeting to aparticular location or cell type, increasing interaction with silencingpathway enzymes, increasing release from endosomes) as an expressioninhibitor.

III. Modified Polynucleotide Agents of the Invention

In one embodiment, the nucleotides of a polynucleotide agent, e.g., anantisense polynucleotide agent, of the invention are un-modified, and donot comprise, e.g., chemical modifications and/or conjugations known inthe art and described herein. In another embodiment, at least one of thenucleotides of a polynucleotide agent of the invention is chemicallymodified to enhance stability or other beneficial characteristics. Incertain embodiments of the invention, substantially all of thenucleotides of a polynucleotide agent of the invention are modified. Inother embodiments of the invention, all of the nucleotides of apolynucleotide agent of the invention are modified. Polynucleotideagents of the invention in which “substantially all of the nucleotidesare modified” are largely but not wholly modified and can include notmore than 5, 4, 3, 2, or 1 unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by standard methods known in the art as further discussedbelow, e.g., solution-phase or solid-phase organic synthesis or both,e.g., by use of an automated DNA synthesizer, such as are commerciallyavailable from, for example, Biosearch, Applied Biosystems®, Inc.Well-established methods for the synthesis and/or modification of thenucleic acids featured in the invention are described in, for example,“Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages.

Specific examples of modified nucleotides useful in the embodimentsdescribed herein include, but are not limited to nucleotides containingmodified backbones or no natural internucleoside linkages. Nucleotideshaving modified backbones include, among others, those that do not havea phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modifiednucleotides that do not have a phosphorus atom in their internucleosidebackbone can also be considered to be oligonucleosides. In someembodiments, a modified polynucleotide agent will have a phosphorus atomin its internucleoside backbone.

Modified nucleotide backbones include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified nucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable nucleotide mimetics are contemplated foruse in polynucleotide agents, in which both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an RNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of an RNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, the entire contents of each of which are herebyincorporated herein by reference. Additional PNA compounds suitable foruse in the polynucleotide agents of the invention are described in, forexample, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include polynucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—-[known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂—- and —N(CH₃)—CH₂—CH₂-[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—-] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, thepolynucleotide agents featured herein have morpholino backbonestructures of the above-referenced U.S. Pat. No. 5,034,506.

Modified nucleotides can also contain one or more modified orsubstituted sugar moieties. The polynucleotide agents featured hereincan include one of the following at the 2′-position: OH; F; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl can be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl.Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.

In other embodiments, polynucleotide agents include one of the followingat the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of a polynucleotide, or a groupfor improving the pharmacodynamic properties of a polynucleotide agent,and other substituents having similar properties. In some embodiments,the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplarymodification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples herein below,and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-O—CH₃) also referred to as2′-OMe, 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F) Similarmodifications can also be made at other positions on a nucleotide of apolynucleotide agent, particularly the 3′ position of the sugar on the3′ terminal nucleotide. Polynucleotide agents can also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative U.S. patents that teach the preparation of suchmodified sugar structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, certain of which are commonly owned with theinstant application. The entire contents of each of the foregoing arehereby incorporated herein by reference.

Additional nucleotides having modified or substituted sugar moieties foruse in the polynucleotide agents of the invention include nucleotidescomprising a bicyclic sugar. A “bicyclic sugar” is a furanosyl ringmodified by the bridging of two atoms. A“bicyclic nucleoside” (“BNA”) isa nucleoside having a sugar moiety comprising a bridge connecting twocarbon atoms of the sugar ring, thereby forming a bicyclic ring system.In certain embodiments, the bridge connects the 4′-carbon and the2′-carbon of the sugar ring. Thus, in some embodiments a polynucleotideagent may include one or more locked nucleic acids. A “locked nucleicacid” (“LNA”) is a nucleotide having a modified ribose moiety in whichthe ribose moiety comprises an extra bridge connecting the 2′ and 4′carbons. In other words, an LNA is a nucleotide comprising a bicyclicsugar moiety comprising a 4′-CH₂—O-2′ bridge. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to polynucleotide agents has been shown toincrease polynucleotide agent stability in serum, and to reduceoff-target effects (Elmen, J. et al., (2005) Nucleic Acids Research33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843;Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Examples of bicyclic nucleosides for use in the polynucleotides of theinvention include without limitation nucleosides comprising a bridgebetween the 4′ and the 2′ ribosyl ring atoms. In certain embodiments,the polynucleotide agents of the invention include one or more bicyclicnucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′bridged bicyclic nucleosides, include but are not limited to4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA);4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No.7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S.Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g.,U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. PatentPublication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672);4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem.,2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 8,278,426). The entire contents of each of theforegoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

In one particular embodiment of the invention, a polynucleotide agentcan include one or more constrained ethyl nucleotides. As used herein, a“constrained ethyl nucleotide” or “cEt” is a locked nucleic acidcomprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.In one embodiment, a constrained ethyl nucleotide is in an Sconformation and is referred to as an “S-constrained ethyl nucleotide”or “S-cEt.”

Modified nucleotides included in the polynucleotide agents of theinvention can also contain one or more sugar mimetics. For example, thepolynucleotide agent may include a “modified tetrahydropyran nucleotide”or “modified THP nucleotide.” A “modified tetrahydropyran nucleotide”has a six-membered tetrahydropyran “sugar” substituted in for thepentofuranosyl residue in normal nucleotides (a sugar surrogate).Modified THP nucleotides include, but are not limited to, what isreferred to in the art as hexitol nucleic acid (HNA), anitol nucleicacid (ANA), manitol nucleic acid (MNA) (see, e.g., Leumann, Bioorg. Med.Chem., 2002, 10, 841-854), or fluoro HNA (F-HNA).

In some embodiments of the invention, sugar surrogates comprise ringshaving more than 5 atoms and more than one heteroatom. For examplenucleotides comprising morpholino sugar moieties and their use inoligomeric compounds has been reported (see for example: Braasch et al.,Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685;5,166,315; 5,185,444; and 5,034,506). Morpholinos may be modified, forexample by adding or altering various substituent groups from the abovemorpholino structure. Such sugar surrogates are referred to herein as“modified morpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH2-O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, compound, e.g., antisense compounds, compriseone or more modified cyclohexenyl nucleosides, which is a nucleosidehaving a six-membered cyclohexenyl in place of the pentofuranosylresidue in naturally occurring nucleosides. Modified cyclohexenylnucleosides include, but are not limited to those described in the art(see for example commonly owned, published PCT Application WO2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem.Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters,2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007,129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids,2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research,2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, SectionF: Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety).

A polynucleotide agent can also include nucleobase modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases such as deoxy-thymine(dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines andguanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in “Modified Nucleosides in Biochemistry,” Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, antisensepolynucleotide agent Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theagents featured in the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., antisense polynucleotide agent Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and areexemplary base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

One or more of the nucleotides of a polynucleotide agent of theinvention may also include a hydroxymethyl substituted nucleotide. A“hydroxymethyl substituted nucleotide” is an acyclic2′-3′-seco-nucleotide, also referred to as an “unlocked nucleic acid”(“UNA”) modification. Representative U.S. publications that teach thepreparation of UNA include, but are not limited to, U.S. Pat. No.8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922;and 2011/0313020, the entire contents of each of which are herebyincorporated herein by reference.

Additional modifications which may potentially stabilize the ends ofpolynucleotide agents can includeN-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3′-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in US Patent PublicationNo. 2012/0142101.

Any of the polynucleotide agents of the invention may be optionallyconjugated with a GalNAc derivative ligand, as described in Section IV,below.

As described in more detail below, an agent that contains conjugationsof one or more carbohydrate moieties to a polynucleotide agent canoptimize one or more properties of the agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of thepolynucleotide agent. For example, the ribose sugar of one or moreribonucleotide subunits of an agent can be replaced with another moiety,e.g., a non-carbohydrate (preferably cyclic) carrier to which isattached a carbohydrate ligand. A ribonucleotide subunit in which theribose sugar of the subunit has been so replaced is referred to hereinas a ribose replacement modification subunit (RRMS). A cyclic carriermay be a carbocyclic ring system, i.e., all ring atoms are carbon atoms,or a heterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The polynucleotide agents may be conjugated to a ligand via a carrier,wherein the carrier can be cyclic group or acyclic group; preferably,the cyclic group is selected from pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,[1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and and decalin; preferably, the acyclic group isselected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the polynucleotide agent for use in themethods of the invention is an agent selected from the group of agentslisted in Tables 3 and 4. These agents may further comprise a ligand, asdescribed in Section IV, below.

A. Polynucleotide Agents Comprising Motifs

In certain embodiments of the invention, at least one of the contiguousnucleotides of the polynucleotide agents, e.g., the antisensepolynucleotide agents, of the invention may be a modified nucleotide. Inone embodiment, the modified nucleotide comprises one or more modifiedsugars. In other embodiments, the modified nucleotide comprises one ormore modified nucleobases. In yet other embodiments, the modifiednucleotide comprises one or more modified internucleoside linkages. Insome embodiments, the modifications (sugar modifications, nucleobasemodifications, and/or linkage modifications) define a pattern or motif.In one embodiment, the patterns of modifications of sugar moieties,internucleoside linkages, and nucleobases are each independent of oneanother.

Polynucleotide agents having modified oligonucleotides arranged inpatterns, or motifs may, for example, confer to the agents propertiessuch as enhanced inhibitory activity, increased binding affinity for atarget nucleic acid, or resistance to degradation by in vivo nucleases.For example, such agents may contain at least one region modified so asto confer increased resistance to nuclease degradation, increasedcellular uptake, increased binding affinity for the target nucleic acid,and/or increased inhibitory activity. A second region of such agents mayoptionally serve as a substrate for the cellular endonuclease RNase H,which cleaves the RNA strand of an RNA:DNA duplex.

An exemplary polynucleotide agent having modified oligonucleotidesarranged in patterns, or motifs is a gapmer. In a “gapmer”, an internalregion or “gap” having a plurality of linked nucleotides that supportsRNaseH cleavage is positioned between two external flanking regions or“wings” having a plurality of linked nucleotides that are chemicallydistinct from the linked nucleotides of the internal region. The gapsegment generally serves as the substrate for endonuclease cleavage,while the wing segments comprise modified nucleotides.

The three regions of a gapmer motif (the 5 ‘-wing, the gap, and the3’-wing) form a contiguous sequence of nucleotides and may be describedas “X-Y-Z”, wherein “X” represents the length of the 5-wing, “Y”represents the length of the gap, and “Z” represents the length of the3′-wing. In one embodiment, a gapmer described as “X-Y-Z” has aconfiguration such that the gap segment is positioned immediatelyadjacent to each of the 5′ wing segment and the 3′ wing segment. Thus,no intervening nucleotides exist between the 5′ wing segment and gapsegment, or the gap segment and the 3′ wing segment. Any of thecompounds, e.g., antisense compounds, described herein can have a gapmermotif. In some embodiments, X and Z are the same, in other embodimentsthey are different.

In certain embodiments, the regions of a gapmer are differentiated bythe types of modified nucleotides in the region. The types of modifiednucleotides that may be used to differentiate the regions of a gapmer,in some embodiments, include β-D-ribonucleotides,(3-D-deoxyribonucleotides, 2′-modified nucleotides, e.g., 2′-modifiednucleotides (e.g., 2′-MOE, and 2′-O—CH3), and bicyclic sugar modifiednucleotides (e.g., those having a 4′-(CH2)n-O-2′ bridge, where n=1 orn=2).

In one embodiment, at least some of the modified nucleotides of each ofthe wings may differ from at least some of the modified nucleotides ofthe gap. For example, at least some of the modified nucleotides of eachwing that are closest to the gap (the 3 ‘-most nucleotide of the 5’-wingand the 5′-most nucleotide of the 3-wing) differ from the modifiednucleotides of the neighboring gap nucleotides, thus defining theboundary between the wings and the gap. In certain embodiments, themodified nucleotides within the gap are the same as one another. Incertain embodiments, the gap includes one or more modified nucleotidesthat differ from the modified nucleotides of one or more othernucleotides of the gap.

The length of the 5′-wing (X) of a gapmer may be 1 to 6 nucleotides inlength, e.g., 2 to 6,2 to 5,3 to 6,3 to 5, 1 to 5, 1 to 4, 1 to 3,2 to 4nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides in length.

The length of the 3′-wing (Z) of a gapmer may be 1 to 6 nucleotides inlength, e.g., 2 to 6, 2-5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides inlength.

The length of the gap (Y) of a gapmer may be 5 to 14 nucleotides inlength, e.g., 5 to 13,5 to 12,5 to 11,5 to 10,5 to 9,5 to 8,5 to 7,5 to6,6 to 14,6 to 13,6 to 12,6 to 11,6 to 10, 6 to 9,6 to 8, 6 to 7,7 to14,7 to 13,7 to 12,7 to 11,7 to 10,7 to 9,7 to 8, 8 to 14, 8 to 13,8 to12,8 to 11,8 to 10,8 to 9,9 to 14,9 to 13,9 to 12,9 to 11,9 to 10, 10 to14, 10 to 13, 10 to 12, 10 to 11, 11 to 14, 11 to 13, 11 to 12, 12 to14, 12 to 13, or 13 to 14 nucleotides in length, e.g., 5, 6, 7, 8, 9,10, 11, 12, 13, or 14 nucleotides in length.

In some embodiments of the invention X consists of 2, 3, 4, 5 or 6nucleotides, Y consists of 7, 8, 9, 10, 11, or 12 nucleotides, and Zconsists of 2, 3, 4, 5 or 6 nucleotides. Such gapmers include (X-Y-Z)2-7-2, 2-7-3, 2-7-4, 2-7-5, 2-7-6, 3-7-2, 3-7-3, 3-7-4, 3-7-5, 3-7-6,4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4,6-7-5, 6-7-6, 3-7-3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6,5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6-7-6, 2-8-2, 2-8-3,2-8-4, 2-8-5, 2-8-6, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-8-6, 4-8-3, 4-8-4,4-8-5, 4-8-6, 5-8-3, 5-8-4, 5-8-5, 5-8-6, 6-8-3, 6-8-4, 6-8-5, 6-8-6,2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-9-6, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-6,4-9-3, 4-9-4, 4-9-5, 4-9-6, 5-9-3, 5-9-4, 5-9-5, 5-9-6, 6-9-3, 6-9-4,6-9-5, 6-9-6, 2-10-2, 2-10-3, 2-10-4, 2-10-5, 2-10-6, 3-10-2, 3-10-3,3-10-4, 3-10-5, 3-10-6, 4-10-3, 4-10-4, 4-10-5, 4-10-6, 5-10-3, 5-10-4,5-10-5, 5-10-6, 6-10-3, 6-10-4, 6-10-5, 6-10-6, 2-11-2, 2-11-3, 2-11-4,2-11-5, 2-11-6, 3-11-2, 3-11-3, 3-11-4, 3-11-5, 3-11-6, 4-11-3, 4-11-4,4-11-5, 4-11-6, 5-11-3, 5-11-4, 5-11-5, 5-11-6, 6-11-3, 6-11-4, 6-11-5,6-11-6, 2- 12-2, 2-12-3, 2-12-4, 2-12-5, 2-12-6, 3-12-2, 3-12-3, 3-12-4,3-12-5, 3-12-6, 4-12-3, 4-12-4, 4-12-5, 4-12-6, 5-12-3, 5-12-4, 5-12-5,5-12-6, 6-12-3, 6-12-4, 6-12-5, or 6-12-6.

In some embodiments of the invention, polynucleotide agents targetingHAO1 include a 5-10-5 gapmer motif. In other embodiments of theinvention, polynucleotide agents targeting HAO1 include a 4-10-4 gapmermotif. In another embodiment of the invention, polynucleotide agentstargeting HAO1 include a 3-10-3 gapmer motif. In yet other embodimentsof the invention, polynucleotide agents targeting HAO1 include a 2-10-2gapmer motif.

The 5′-wing and/or 3′-wing of a gapmer may independently include 1-6modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

In some embodiment, the 5′-wing of a gapmer includes at least onemodified nucleotide. In one embodiment, the 5′-wing of a gapmercomprises at least two modified nucleotides. In another embodiment, the5′-wing of a gapmer comprises at least three modified nucleotides. Inyet another embodiment, the 5′-wing of a gapmer comprises at least fourmodified nucleotides. In another embodiment, the 5′-wing of a gapmercomprises at least five modified nucleotides. In certain embodiments,each nucleotide of the 5′-wing of a gapmer is a modified nucleotide.

In some embodiments, the 3′-wing of a gapmer includes at least onemodified nucleotide. In one embodiment, the 3′-wing of a gapmercomprises at least two modified nucleotides. In another embodiment, the3′-wing of a gapmer comprises at least three modified nucleotides. Inyet another embodiment, the 3′-wing of a gapmer comprises at least fourmodified nucleotides. In another embodiment, the 3′-wing of a gapmercomprises at least five modified nucleotides. In certain embodiments,each nucleotide of the 3′-wing of a gapmer is a modified nucleotide.

In certain embodiments, the regions of a gapmer are differentiated bythe types of sugar moieties of the nucleotides. In one embodiment, thenucleotides of each distinct region comprise uniform sugar moieties. Inother embodiments, the nucleotides of each distinct region comprisedifferent sugar moieties. In certain embodiments, the sugar nucleotidemodification motifs of the two wings are the same as one another. Incertain embodiments, the sugar nucleotide modification motifs of the5′-wing differs from the sugar nucleotide modification motif of the3′-wing.

The 5′-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1,2, 3, 4, 5, or 6 modified nucleotides.

In one embodiment, at least one modified nucleotide of the 5′-wing of agapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide,or an LNA. In another embodiment, the 5′-wing of a gapmer includes 2, 3,4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide ofthe 5′-wing of a gapmer is a bicyclic nucleotide.

In one embodiment, the 5′-wing of a gapmer includes at least 1, 2, 3, 4,or 5 constrained ethyl nucleotides. In some embodiments, each nucleotideof the 5′-wing of a gapmer is a constrained ethyl nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one LNAnucleotide. In another embodiment, the 5′-wing of a gapmer includes 2,3, 4, or 5 LNA nucleotides. In other embodiments, each nucleotide of the5′-wing of a gapmer is an LNA nucleotide.

In certain embodiments, at least one modified nucleotide of the 5′-wingof a gapmer is a non-bicyclic modified nucleotide, e.g., a 2‘-substituted nucleotide. A “2’-substituted nucleotide” is a nucleotidecomprising a modification at the 2′-position which is other than H orOH, such as a 2′-OMe nucleotide, or a 2′-MOE nucleotide. In oneembodiment, the 5′-wing of a gapmer comprises 2, 3, 4, or 5 2‘-substituted nucleotides. In one embodiment, each nucleotide of the5’-wing of a gapmer is a 2′-substituted nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one 2′-OMenucleotide. In one embodiment, the 5′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-OMe nucleotides. In one embodiment, each of thenucleotides of the 5′-wing of a gapmer comprises a 2′-OMe nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one 2′-MOEnucleotide. In one embodiment, the 5′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-MOE nucleotides. In one embodiment, each of thenucleotides of the 5′-wing of a gapmer comprises a 2′-MOE nucleotide.

In certain embodiments, the 5′-wing of a gapmer comprises at least one2′-deoxynucleotide. In certain embodiments, each nucleotide of the5′-wing of a gapmer is a 2′-deoxynucleotide. In a certain embodiments,the 5′-wing of a gapmer comprises at least one ribonucleotide. Incertain embodiments, each nucleotide of the 5′-wing of a gapmer is aribonucleotide.

The 3′-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1,2, 3, 4, 5, or 6 modified nucleotides.

In one embodiment, at least one modified nucleotide of the 3′-wing of agapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide,or an LNA. In another embodiment, the 3′-wing of a gapmer includes 2, 3,4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide ofthe 3′-wing of a gapmer is a bicyclic nucleotide.

In one embodiment, the 3′-wing of a gapmer includes at least oneconstrained ethyl nucleotide. In another embodiment, the 3′-wing of agapmer includes 2, 3, 4, or 5 constrained ethyl nucleotides. In someembodiments, each nucleotide of the 3′-wing of a gapmer is a constrainedethyl nucleotide.

In one embodiment, the 3′-wing of a gapmer comprises at least one LNAnucleotide. In another embodiment, the 3′-wing of a gapmer includes 2,3, 4, or 5 LNA nucleotides. In other embodiments, each nucleotide of the3′-wing of a gapmer is an LNA nucleotide.

In certain embodiments, at least one modified nucleotide of the 3′-wingof a gapmer is a non-bicyclic modified nucleotide, e.g., a 2‘-substituted nucleotide. In one embodiment, the 3’-wing of a gapmercomprises 2, 3, 4, or 5 2 ‘-substituted nucleotides. In one embodiment,each nucleotide of the 3’-wing of a gapmer is a 2 ‘-substitutednucleotide.

In one embodiment, the 3’-wing of a gapmer comprises at least one 2′-OMenucleotide. In one embodiment, the 3′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-OMe nucleotides. In one embodiment, each of thenucleotides of the 3′-wing of a gapmer comprises a 2′-OMe nucleotide.

In one embodiment, the 3′-wing of a gapmer comprises at least one 2′-MOEnucleotide. In one embodiment, the 3′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-MOE nucleotides. In one embodiment, each of thenucleotides of the 3′-wing of a gapmer comprises a 2′-MOE nucleotide.

In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-deoxynucleotide. In certain embodiments, each nucleotide of the3′-wing of a gapmer is a 2′-deoxynucleotide. In a certain embodiments,the 3′-wing of a gapmer comprises at least one ribonucleotide. Incertain embodiments, each nucleotide of the 3′-wing of a gapmer is aribonucleotide.

The gap of a gapmer may include 5-14 modified nucleotides, e.g., 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 modified nucleotides.

In one embodiment, the gap of a gapmer comprises at least one5-methylcytosine. In one embodiment, the gap of a gapmer comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 5-methylcytosines. Inone embodiment, all of the nucleotides of the the gap of a gapmer are5-methylcytosines.

In one embodiment, the gap of a gapmer comprises at least one2′-deoxynucleotide. In one embodiment, the gap of a gapmer comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 2′-deoxynucleotides. Inone embodiment, all of the nucleotides of the the gap of a gapmer are2′-deoxynucleotides.

A gapmer may include one or more modified internucleotide linkages. Insome embodiments, a gapmer includes one or more phosphodiesterinternucleotide linkages. In other embodiments, a gapmer includes one ormore phosphorothioate internucleotide linkages.

In one embodiment, each nucleotide of a 5′-wing of a gapmer are linkedvia a phosphorothioate internucleotide linkage. In another embodiment,each nucleotide of a 3′-wing of a gapmer are linked via aphosphorothioate internucleotide linkage. In yet another embodiment,each nucleotide of a gap segment of a gapmer is linked via aphosphorothioate internucleotide linkage. In one embodiment, all of thenucleotides in a gapmer are linked via phosphorothioate internucleotidelinkages.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides and a 3′-wing segment comprising 5 nucleotides.

In another embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fournucleotides and a 3′-wing segment comprising four nucleotides.

In another embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threenucleotides and a 3′-wing segment comprising three nucleotides.

In another embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twonucleotides and a 3′-wing segment comprising two nucleotides.

In one embodiment, each nucleotide of a 5-wing flanking a gap segment of10 2′-deoxyribonucleotides comprises a modified nucleotide. In anotherembodiment, each nucleotide of a 3-wing flanking a gap segment of 102′-deoxyribonucleotides comprises a modified nucleotide. In oneembodiment, each of the modified 5′-wing nucleotides and each of themodified 3′-wing nucleotides comprise a 2′-sugar modification. In oneembodiment, the 2′-sugar modification is a 2′-OMe modification. Inanother embodiment, the 2′-sugar modification is a 2′-MOE modification.In one embodiment, each of the modified 5′-wing nucleotides and each ofthe modified 3′-wing nucleotides comprise a bicyclic nucleotide. In oneembodiment, the bicyclic nucleotide is a constrained ethyl nucleotide.In another embodiment, the bicyclic nucleotide is an LNA nucleotide. Inone embodiment, each cytosine in a polynucleotide agent targeting anHAO1 gene is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising five nucleotides comprising a 2′ OMe modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine. Inone embodiment, the agent further comprises a ligand.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising five nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine. Inone embodiment, the agent further comprises a ligand.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fiveconstrained ethyl nucleotides and a 3′-wing segment comprising fiveconstrained ethyl nucleotides, wherein each internucleotide linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fiveLNA nucleotides and a 3′-wing segment comprising five LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fournucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising four nucleotides comprising a 2′ OMe modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fournucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising four nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fourconstrained ethyl nucleotides and a 3′-wing segment comprising fourconstrained ethyl nucleotides, wherein each internucleotide linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fourLNA nucleotides and a 3′-wing segment comprising four LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threenucleotides comprising a 2′ OMe modification and a 3′-wing segmentcomprising three nucleotides comprising a 2′ OMe modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threenucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising three nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threeconstrained ethyl nucleotides and a 3′-wing segment comprising threeconstrained ethyl nucleotides, wherein each internucleotide linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threeLNA nucleotides and a 3′-wing segment comprising three LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twonucleotides comprising a 2′ OMe modification and a 3′-wing segmentcomprising two nucleotides comprising a 2′ OMe modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twonucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising two nucleotides comprising a 2′MOE modification, wherein eachinternucleotide linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twoconstrained ethyl nucleotides and a 3′-wing segment comprising twoconstrained ethyl nucleotides, wherein each internucleotide linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, a polynucleotide agent targeting an HAO1 genecomprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising two LNAnucleotides and a 3′-wing segment comprising two LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

Further gapmer designs suitable for use in the agents, compositions, andmethods of the invention are disclosed in, for example, U.S. Pat. Nos.7,687,617 and 8,580,756; U.S. Patent Publication Nos. 20060128646,20090209748, 20140128586, 20140128591, 20100210712, and 20080015162A1;and International Publication No. WO 2013/159108, the entire content ofeach of which are incorporated herein by reference.

IV. Polynucleotide Agents Conjugated to Ligands

Another modification of the polynucleotide agents of the inventioninvolves chemically linking to the agent one or more ligands, moietiesor conjugates that enhance the activity, cellular distribution orcellular uptake of the polynucleotide agent. Such moieties include butare not limited to lipid moieties such as a cholesterol moiety(Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556),cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994,4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med.Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of a polynucleotide agent into which it is incorporated. Inpreferred embodiments a ligand provides an enhanced affinity for aselected target, e.g., molecule, cell or cell type, compartment, e.g., acellular or organ compartment, tissue, organ or region of the body, as,e.g., compared to a species absent such a ligand. Preferred ligands willnot take part in hybridization of a polynucleotide agent to the targetedmRNA.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the polynucleotide agent into the cell, for example, bydisrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

In some embodiments, a ligand attached to a polynucleotide agent asdescribed herein acts as a pharmacokinetic modulator (PK modulator). PKmodulators include lipophiles, bile acids, steroids, phospholipidanalogues, peptides, protein binding agents, PEG, vitamins etc.Exemplary PK modulators include, but are not limited to, cholesterol,fatty acids, cholic acid, lithocholic acid, dialkylglycerides,diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen,vitamin E, biotin etc. Oligonucleotides that comprise a number ofphosphorothioate linkages are also known to bind to serum protein, thusshort oligonucleotides, e.g., oligonucleotides of about 5 bases, 10bases, 15 bases or 20 bases, comprising multiple of phosphorothioatelinkages in the backbone are also amenable to the present invention asligands (e.g. as PK modulating ligands). In addition, aptamers that bindserum components (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated polynucleotides of the invention may be synthesized bythe use of a polynucleotide that bears a pendant reactive functionality,such as that derived from the attachment of a linking molecule onto theoligonucleotide (described below). This reactive polynucleotide may bereacted directly with commercially-available ligands, ligands that aresynthesized bearing any of a variety of protecting groups, or ligandsthat have a linking moiety attached thereto.

The polynucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems® (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other polynucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated polynucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, thepolynucleotides and polynucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the polynucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to polynucleotideagents can affect pharmacokinetic distribution of the agent, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 5). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 6) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 7) and theDrosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 8) havebeen found to be capable of functioning as delivery peptides. A peptideor peptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to anantisens epolynucleotide agent via an incorporated monomer unit for celltargeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide,or RGD mimic A peptide moiety can range in length from about 5 aminoacids to about 40 amino acids. The peptide moieties can have astructural modification, such as to increase stability or directconformational properties. Any of the structural modifications describedbelow can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, apolynucleotide agent further comprises a carbohydrate. The carbohydrateconjugated agents are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein (see, e.g., Prakash, et al. (2014) Nuc Acid Res doi10.1093/nar/gku531). As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In one embodiment, themonosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

from the group consisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to

when one of X or Y is an oligonucleotide, the other is a hydrogen.

when one of X or Y is an oligonucleotide, the other is a hydrogen; orboth X and Y are oligonucleotides

wherein Y is O or S and n is 1-6.

wherein Y═O or S. n is 1-6, R is hydrogen or nucleic acid, R′ is nucleicacid.

wherein Y is O or S and n is 1-6.

when one of X or Y is an oligonucleotide, the other is a hydrogen; orboth X and Y are oligonucleotides

wherein X is O or S.

In certain embodiments of the invention, the GalNAc or GalNAc derivativeis attached to a polynucleotide agent of the invention via a monovalentlinker. In some embodiments, the GalNAc or GalNAc derivative is attachedto a polynucleotide of the invention via a bivalent linker. In yet otherembodiments of the invention, the GalNAc or GalNAc derivative isattached to a polynucleotide of the invention via a trivalent linker.

In one embodiment, the polynucleotide agents of the invention compriseone GalNAc or GalNAc derivative attached to the iRNA agent. In anotherembodiment, the polynucleotide agents of the invention comprise aplurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, eachindependently attached to a plurality of nucleotides of thepolynucleotide agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of apolynucleotide agent of the invention are part of one larger moleculeconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming ahairpin loop comprising, a plurality of unpaired nucleotides, eachunpaired nucleotide within the hairpin loop may independently comprise aGalNAc or GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in thepresent invention include those described in PCT Publication Nos. WO2014/179620 and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an polynucleotide agent with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular polynucleotide agent moiety andparticular targeting agent one can look to methods described herein. Forexample, a candidate can be evaluated by incubation with dithiothreitol(DTT), or other reducing agent using reagents know in the art, whichmimic the rate of cleavage which would be observed in a cell, e.g., atarget cell. The candidates can also be evaluated under conditions whichare selected to mimic blood or serum conditions. In one, candidatecompounds are cleaved by at most about 10% in the blood. In otherembodiments, useful candidate compounds are degraded at least about 2,4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in thecell (or under in vitro conditions selected to mimic intracellularconditions) as compared to blood (or under in vitro conditions selectedto mimic extracellular conditions). The rate of cleavage of candidatecompounds can be determined using standard enzyme kinetics assays underconditions chosen to mimic intracellular media and compared toconditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, a polynucleotide agent of the invention is conjugatedto a carbohydrate through a linker. Non-limiting examples ofpolynucleotide agent carbohydrate conjugates with linkers of thecompositions and methods of the invention include, but are not limitedto,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a polynucleotide agent of the invention is conjugatedto a bivalent or trivalent branched linker selected from the group ofstructures shown in any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), CC or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with polynucleotide agents for inhibitingthe expression of a target gene, such as those of formula (XXXVI):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within a polynucleotide agent. The present inventionalso includes polynucleotide agents that are chimeric compounds.

“Chimeric” polynucleotide agents or “chimeras,” in the context of thisinvention, are polynucleotide agent compounds, which contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of a polynucleotide agent. Thesepolynucleotide agents typically contain at least one region wherein theRNA is modified so as to confer upon the polynucleotide agent increasedresistance to nuclease degradation, increased cellular uptake, and/orincreased binding affinity for the target nucleic acid. An additionalregion of the polynucleotide agent can serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of polynucleotide agentinhibition of gene expression. Consequently, comparable results canoften be obtained with shorter polynucleotide agents when chimericpolynucleotide agents are used, compared to phosphorothioate deoxypolynucleotide agents hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

In certain instances, the nucleotide of a polynucleotide agent can bemodified by a non-ligand group. A number of non-ligand molecules havebeen conjugated to polynucleotide agents in order to enhance theactivity, cellular distribution or cellular uptake of the polynucleotideagent, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res.Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett.,1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl.Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such RNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of an RNAs bearingan aminolinker at one or more positions of the sequence. The amino groupis then reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction can beperformed either with the RNA still bound to the solid support orfollowing cleavage of the RNA, in solution phase. Purification of theRNA conjugate by HPLC typically affords the pure conjugate.

V. Delivery of a Polynucleotide Agent of the Invention

The delivery of a polynucleotide agent of the invention, e.g., anantisense polynucleotide agent, to a cell e.g., a cell within a subject,such as a human subject (e.g., a subject in need thereof, such as asubject having an HAO1-associated disease) can be achieved in a numberof different ways. For example, delivery may be performed by contactinga cell with a polynucleotide agent of the invention either in vitro orin vivo. In vivo delivery may also be performed directly byadministering a composition comprising a polynucleotide agent to asubject.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with a polynucleotide agent of theinvention (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol.2(5):139-144 and WO94/02595, which are incorporated herein by referencein their entireties). For in vivo delivery, factors to consider in orderto deliver a polynucleotide agent include, for example, biologicalstability of the delivered molecule, prevention of non-specific effects,and accumulation of the delivered molecule in the target tissue. Thenon-specific effects of a polynucleotide agent can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the polynucleotide agent to be administered. Severalstudies have shown successful knockdown of gene products when apolynucleotide agent is administered locally. For example, intraoculardelivery of a VEGF antisense polynucleotide agent by intravitrealinjection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina24:132-138) and subretinal injections in mice (Reich, S J., et al (2003)Mol. Vis. 9:210-216) were both shown to prevent neovascularization in anexperimental model of age-related macular degeneration. In addition,direct intratumoral injection of an antisense polynucleotide agent inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering a polynucleotide agent systemically for the treatment of adisease, the agent can be modified or alternatively delivered using adrug delivery system; both methods act to prevent the rapid degradationof the polynucleotide agent by endo- and exo-nucleases in vivo.Modification of the agent or the pharmaceutical carrier can also permittargeting of the polynucleotide agent composition to the target tissueand avoid undesirable off-target effects. Polynucleotide agent can bemodified by chemical conjugation to lipophilic groups such ascholesterol to enhance cellular uptake and prevent degradation. In analternative embodiment, the polynucleotide agent can be delivered usingdrug delivery systems such as a nanoparticle, a dendrimer, a polymer,liposomes, or a cationic delivery system. Positively charged cationicdelivery systems facilitate binding of a polynucleotide agent molecule(negatively charged) and also enhance interactions at the negativelycharged cell membrane to permit efficient uptake of a polynucleotideagent by the cell. Cationic lipids, dendrimers, or polymers can eitherbe bound to a polynucleotide agent, or induced to form a vesicle ormicelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release129(2):107-116) that encases a polynucleotide agent. The formation ofvesicles or micelles further prevents degradation of the polynucleotideagent when administered systemically. Methods for making andadministering cationic-polynucleotide agent complexes are well withinthe abilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of polynucleotide agents include DOTAP (Sorensen, D R., et al(2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solidnucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther.12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091),polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epubahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659),Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), andpolyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans.35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In someembodiments, a polynucleotide agent forms a complex with cyclodextrinfor systemic administration. Methods for administration andpharmaceutical compositions of polynucleotide agents and cyclodextrinscan be found in U.S. Pat. No. 7,427,605, which is herein incorporated byreference in its entirety.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the polynucleotide agents, e.g., theantisense polynucleotide agents, of the invention. In one embodiment,provided herein are pharmaceutical compositions containing apolynucleotide agent, as described herein, and a pharmaceuticallyacceptable carrier.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum components, such as serum albumin, HDL and LDL; and(22) other non-toxic compatible substances employed in pharmaceuticalformulations.

The pharmaceutical compositions containing the polynucleotide agents areuseful for treating a disease or disorder associated with the expressionor activity of an HAO1 gene, e.g. an HAO1-associated disease. Suchpharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by subcutaneous (SC) orintravenous (IV) delivery. Another example is compositions that areformulated for direct delivery into the brain parenchyma, e.g., byinfusion into the brain, such as by continuous pump infusion. Thepharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of an HAO1 gene. In general, asuitable dose of a polynucleotide agent of the invention will be in therange of about 0.001 to about 200.0 milligrams per kilogram body weightof the recipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. For example, the polynucleotide agent canbe administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg,about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kgper single dose.

For example, the polynucleotide agent may be administered at a dose ofabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, orabout 50 mg/kg. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

In another embodiment, the polynucleotide agent is administered at adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

For example, the polynucleotide agent may be administered at a dose ofabout 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

In another embodiment, the polynucleotide agent is administered at adose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about1 to about 50 mg/mg, about 1.5 to about 50 mg/kgb, about 2 to about 50mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, thepolynucleotide agent is administered at a dose of about 10 mg/kg toabout 30 mg/kg. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

For example, subjects can be administered, e.g., subcutaneously orintravenously, a single therapeutic amount of polynucleotide agent, suchas about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325,0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6,0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875,0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4,3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28,28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In some embodiments, subjects are administered, e.g., subcutaneously orintravenously, multiple doses of a therapeutic amount of polynucleotideagent, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25,0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525,0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8,0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9,9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26,26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Amulti-dose regimen may include administration of a therapeutic amount ofpolynucleotide agent daily, such as for two days, three days, four days,five days, six days, seven days, or longer.

In other embodiments, subjects are administered, e.g., subcutaneously orintravenously, a repeat dose of a therapeutic amount of polynucleotideagent, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25,0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525,0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8,0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9,9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26,26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Arepeat-dose regimen may include administration of a therapeutic amountof polynucleotide agent on a regular basis, such as every other day,every third day, every fourth day, twice a week, once a week, everyother week, or once a month.

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered once daily, or thepharmaceutical composition can be administered as two, three, or moresub-doses at appropriate intervals throughout the day or even usingcontinuous infusion or delivery through a controlled releaseformulation. In that case, the polynucleotide agent contained in eachsub-dose must be correspondingly smaller in order to achieve the totaldaily dosage. The dosage unit can also be compounded for delivery overseveral days, e.g., using a conventional sustained release formulationwhich provides sustained release of the polynucleotide agent over aseveral day period. Sustained release formulations are well known in theart and are particularly useful for delivery of agents at a particularsite, such as could be used with the agents of the present invention. Inthis embodiment, the dosage unit contains a corresponding multiple ofthe daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual polynucleotide agents encompassedby the invention can be made using conventional methodologies or on thebasis of in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as a disorder that wouldbenefit from reduction in the expression of HAO1. Such models can beused for in vivo testing of a polynucleotide agent, as well as fordetermining a therapeutically effective dose. Suitable mouse models mayinclude mutations or deletions in the AGXT or GRHPR genes and are knownin the art (see, e.g., Salido E C, et al. (2006) PNAS 103(48):18249-18254 and Knight J, et al. (2012) Am. J. Physiol. Renal Physiol.302: F688-F693).

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The polynucleotide agent can be delivered in a manner to target aparticular tissue, such as the liver (e.g., the hepatocytes of theliver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the polynucleotide agents featured in the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Polynucleotide agents featured in the invention canbe encapsulated within liposomes or can form complexes thereto, inparticular to cationic liposomes. Alternatively, polynucleotide agentscan be complexed to lipids, in particular to cationic lipids. Suitablefatty acids and esters include but are not limited to arachidonic acid,oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, anacylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM),monoglyceride, diglyceride or pharmaceutically acceptable salt thereof).Topical formulations are described in detail in U.S. Pat. No. 6,747,014,which is incorporated herein by reference.

A. Polynucleotide Agent Formulations Comprising Membranous MolecularAssemblies

A polynucleotide agent for use in the compositions and methods of theinvention can be formulated for delivery in a membranous molecularassembly, e.g., a liposome or a micelle. As used herein, the term“liposome” refers to a vesicle composed of amphiphilic lipids arrangedin at least one bilayer, e.g., one bilayer or a plurality of bilayers.Liposomes include unilamellar and multilamellar vesicles that have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the polynucleotide agent composition. Thelipophilic material isolates the aqueous interior from an aqueousexterior, which typically does not include the polynucleotide agentcomposition, although in some examples, it may. Liposomes are useful forthe transfer and delivery of active ingredients to the site of action.Because the liposomal membrane is structurally similar to biologicalmembranes, when liposomes are applied to a tissue, the liposomal bilayerfuses with bilayer of the cellular membranes. As the merging of theliposome and cell progresses, the internal aqueous contents that includethe polynucleotide agent are delivered into the cell where thepolynucleotide can specifically bind to a target RNA and can mediateantisense inhibition. In some cases the liposomes are also specificallytargeted, e.g., to direct the polynucleotide agent to particular celltypes.

A liposome containing a polynucleotide agent can be prepared by avariety of methods. In one example, the lipid component of a liposome isdissolved in a detergent so that micelles are formed with the lipidcomponent. For example, the lipid component can be an amphipathiccationic lipid or lipid conjugate. The detergent can have a highcritical micelle concentration and may be nonionic. Exemplary detergentsinclude cholate, CHAPS, octylglucoside, deoxycholate, and lauroylsarcosine. The polynucleotide agent preparation is then added to themicelles that include the lipid component. The cationic groups on thelipid interact with the polynucleotide agent and condense around thepolynucleotide agent to form a liposome. After condensation, thedetergent is removed, e.g., by dialysis, to yield a liposomalpreparation of polynucleotide agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also be adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No.5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al.Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci.75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984;Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging polynucleotide agentpreparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550,1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human GeneTher. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J.11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver polynucleotide agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated polynucleotide agents in their internalcompartments from metabolism and degradation (Rosoff, in “PharmaceuticalDosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in the preparation of liposomeformulations are the lipid surface charge, vesicle size and the aqueousvolume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of polynucleotide agent(see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMAand its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration; liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a polynucleotide agent into the skin. In someimplementations, liposomes are used for delivering polynucleotide agenstto epidermal cells and also to enhance the penetration of polynucleotideagenst into dermal tissues, e.g., into skin. For example, the liposomescan be applied topically. Topical delivery of drugs formulated asliposomes to the skin has been documented (see, e.g., Weiner et al.,Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al.,Antiviral Research, 18, 1992, 259-265; Mannino, R. J. andFould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987;Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983;Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855,1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin Such formulationswith polynucleotide agents are useful for treating a dermatologicaldisorder.

Liposomes that include polynucleotide agent can be made highlydeformable. Such deformability can enable the liposomes to penetratethrough pore that are smaller than the average radius of the liposome.For example, transfersomes are a type of deformable liposomes.Transferosomes can be made by adding surface edge activators, usuallysurfactants, to a standard liposomal composition. Transfersomes thatinclude polynucleotide agents can be delivered, for example,subcutaneously by infection in order to deliver polynucleotide agents tokeratinocytes in the skin. In order to cross intact mammalian skin,lipid vesicles must pass through a series of fine pores, each with adiameter less than 50 nm, under the influence of a suitable transdermalgradient. In addition, due to the lipid properties, these transferosomescan be self-optimizing (adaptive to the shape of pores, e.g., in theskin), self-repairing, and can frequently reach their targets withoutfragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The polynucleotide agent for use in the compositions and methods of theinvention can also be provided as micellar formulations. “Micelles” aredefined herein as a particular type of molecular assembly in whichamphipathic molecules are arranged in a spherical structure such thatall the hydrophobic portions of the molecules are directed inward,leaving the hydrophilic portions in contact with the surrounding aqueousphase. The converse arrangement exists if the environment ishydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of thepolynucleotide agent composition, an alkali metal C₈ to C₂₂ alkylsulphate, and a micelle forming compounds. Exemplary micelle formingcompounds include lecithin, hyaluronic acid, pharmaceutically acceptablesalts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract,cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein,monooleates, monolaurates, borage oil, evening of primrose oil, menthol,trihydroxy oxo cholanyl glycine and pharmaceutically acceptable saltsthereof, glycerin, polyglycerin, lysine, polylysine, triolein,polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethersand analogues thereof, chenodeoxycholate, deoxycholate, and mixturesthereof. The micelle forming compounds may be added at the same time orafter addition of the alkali metal alkyl sulphate. Mixed micelles willform with substantially any kind of mixing of the ingredients butvigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe polynucleotide agent composition and at least the alkali metal alkylsulphate. The first micellar composition is then mixed with at leastthree micelle forming compounds to form a mixed micellar composition. Inanother method, the micellar composition is prepared by mixing thepolynucleotide agent composition, the alkali metal alkyl sulphate and atleast one of the micelle forming compounds, followed by addition of theremaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

Polynucleotide agents of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle comprising a lipid layer encapsulating a pharmaceuticallyactive molecule. LNPs typically contain a cationic lipid, a non-cationiclipid, and a lipid that prevents aggregation of the particle (e.g., aPEG-lipid conjugate). LNPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). LNPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 nm toabout 90 nm, and are substantially nontoxic. In addition, the nucleicacids when present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; 6,858,225; 8,158,601; and 8,058,069; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to polynucleotide agent ratio) will be in the range of from about1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, orabout 6:1 to about 9:1. Ranges intermediate to the above recited rangesare also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane(DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-polynucleotide agent nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-polynucleotide agent particle includes 40%2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 polynucleotide agent/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (CO, a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl(MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-polynucleotide agent nanoparticles (i.e., LNP01 particles). Stocksolutions of each in ethanol can be prepared as follows: ND98, 133mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous polynucleotide (e.g., in sodium acetate pH 5) suchthat the final ethanol concentration is about 35-45% and the finalsodium acetate concentration is about 100-300 mM. Lipid-polynucleotideagent nanoparticles typically form spontaneously upon mixing. Dependingon the desired particle size distribution, the resultant nanoparticlemixture can be extruded through a polycarbonate membrane (e.g., 100 nmcut-off) using, for example, a thermobarrel extruder, such as LipexExtruder (Northern Lipids, Inc). In some cases, the extrusion step canbe omitted. Ethanol removal and simultaneous buffer exchange can beaccomplished by, for example, dialysis or tangential flow filtration.Buffer can be exchanged with, for example, phosphate buffered saline(PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1,about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-polynucleotide agent formulations aredescribed in Table 1.

TABLE 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:polynucleotide agent ratioSNALP- 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMA 1dimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:polynucleotideagent ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DPPC/Cholesterol/PEG-cDMA dioxolane (XTC) 57.1/7.1/34.4/1.4lipid:polynucleotide agent ~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:polynucleotide agent ~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:polynucleotide agent ~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 60/7.5/31/1.5, lipid:polynucleotide agent ~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 60/7.5/31/1.5, lipid:polynucleotide agent ~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 50/10/38.5/1.5 Lipid:polynucleotide agent 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100)Lipid:polynucleotide agent 10:1 LNP11(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMGtetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3)Lipid:polynucleotide agent 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- TechG1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1-Lipid:polynucleotide agent 10:1 yl)ethylazanediyl)didodecan-2-ol (TechG1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:polynucleotideagent: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5Lipid:polynucleotide agent: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5Lipid:polynucleotide agent: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:polynucleotide agent: 7:1 LNP17 MC3MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:polynucleotide agent: 10:1LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:polynucleotideagent: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5Lipid:polynucleotide agent: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG50/10/38.5/1.5 Lipid:polynucleotide agent: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:polynucleotide agent: 7:1LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:polynucleotideagent: 10:1DSPC: distearoylphosphatidylcholineDPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, protecting groups can be used. Protecting groupmethodology is well known to those skilled in the art (see, for example,Protective Groups in Organic Synthesis, Green, T. W. et al.,Wiley-Interscience, New York City, 1999). Briefly, protecting groupswithin the context of this invention are any group that reduces oreliminates unwanted reactivity of a functional group. A protecting groupcan be added to a functional group to mask its reactivity during certainreactions and then removed to reveal the original functional group. Insome embodiments an “alcohol protecting group” is used. An “alcoholprotecting group” is any group which decreases or eliminates unwantedreactivity of an alcohol functional group. Protecting groups can beadded and removed using techniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g). Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa2SO4 solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H]−232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A-Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3,[M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR 5=130.2, 130.1 (×2),127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7,29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+Calc. 654.6,Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalpolynucleotide agent concentration in the formulation, as well as theentrapped fraction, is estimated using a dye exclusion assay. A sampleof the formulated polynucleotide agent can be incubated with anRNA-binding dye, such as Ribogreen (Molecular Probes) in the presence orabsence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100.The total polynucleotide agent in the formulation can be determined bythe signal from the sample containing the surfactant, relative to astandard curve. The entrapped fraction is determined by subtracting the“free” polynucleotide agent content (as measured by the signal in theabsence of surfactant) from the total polynucleotide agent content.Percent entrapped polynucleotide agent is typically >85%. For SNALPformulation, the particle size is at least 30 nm, at least 40 nm, atleast 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitablerange is typically about at least 50 nm to about at least 110 nm, aboutat least 60 nm to about at least 100 nm, or about at least 80 nm toabout at least 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which the polynucleotide agents featured inthe invention are administered in conjunction with one or morepenetration enhancer surfactants and chelators. Suitable surfactantsinclude fatty acids and/or esters or salts thereof, bile acids and/orsalts thereof. Suitable bile acids/salts include chenodeoxycholic acid(CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid,dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid,glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Polynucleotide agents featured in the invention can be delivered orally,in granular form including sprayed dried particles, or complexed to formmicro or nanoparticles. Polynucleotide agent complexing agents includepoly-amino acids; polyimines; polyacrylates; polyalkylacrylates,polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins,starches, acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor polynucleotide agents and their preparation are described in detailin U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No.6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver, e.g., when treatinghepatic disorders, e.g., hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and antioxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, LV., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e. g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions ofpolynucleotide agents are formulated as microemulsions. A microemulsioncan be defined as a system of water, oil and amphiphile which is asingle optically isotropic and thermodynamically stable liquid solution(see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, LV., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions aresystems that are prepared by first dispersing an oil in an aqueoussurfactant solution and then adding a sufficient amount of a fourthcomponent, generally an intermediate chain-length alcohol to form atransparent system. Therefore, microemulsions have also been describedas thermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen,LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or polynucleotide agents. Microemulsions have also beeneffective in the transdermal delivery of active components in bothcosmetic and pharmaceutical applications. It is expected that themicroemulsion compositions and formulations of the present inventionwill facilitate the increased systemic absorption of polynucleotideagents from the gastrointestinal tract, as well as improve the localcellular uptake of polynucleotide agents and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the polynucleotide agentsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories--surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

A polynucleotide agent of the invention may be incorporated into aparticle, e.g., a microparticle. Microparticles can be produced byspray-drying, but may also be produced by other methods includinglyophilization, evaporation, fluid bed drying, vacuum drying, or acombination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly polynucleotide agents, to the skin of animals. Most drugsare present in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs cancross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of polynucleotide agentsthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (see e.g., Malmsten, M. Surfactants and polymers in drugdelivery, Informa Health Care, New York, N.Y., 2002; Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); andperfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm.Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption ofpolynucleotide agents through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Suitable chelating agents include butare not limited to disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. etal., Excipient development for pharmaceutical, biotechnology, and drugdelivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al.,J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of polynucleotide agents through the alimentarymucosa (see e.g., Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersincludes, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of polynucleotide agents at the cellularlevel can also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof polynucleotide agents. Examples of commercially availabletransfection reagents include, for example Lipofectamine™ (Invitrogen;Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.),293fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen;Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioated polynucleotide agent in hepatic tissue can be reducedwhen it is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense polynucleotide agent Res. Dev., 1995, 5,115-121; Takakura et al., Antisense polynucleotide agent & Nucl. AcidDrug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more polynucleotide agents and (b) one ormore agents which function by a non-antisense inhibition mechanism andwhich are useful in treating a hemolytic disorder. Examples of suchagents include, but are not limited to an anti-inflammatory agent,anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. Inaddition, other substances commonly used to protect the liver, such assilymarin, can also be used in conjunction with the polynucleotideagents described herein. Other agents useful for treating liver diseasesinclude telbivudine, entecavir, and protease inhibitors such astelaprevir and other disclosed, for example, in Tung et al., U.S.Application Publication Nos. 2005/0148548, 2004/0167116, and2003/0144217; and in Hale et al., U.S. Application Publication No.2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, thepolynucleotide agents featured in the invention can be administered incombination with other known agents effective in treatment ofpathological processes mediated by HAO1 expression. In any event, theadministering physician can adjust the amount and timing ofpolynucleotide agent administration on the basis of results observedusing standard measures of efficacy known in the art or describedherein.

VII. Methods for Inhibiting HAO1 Expression

The present invention provides methods of inhibiting expression of HAO1in a cell. The methods include contacting a cell with a polynucleotideagent of the invention, e.g., an antisense polynucleotide agent, in anamount effective to inhibit expression of the HAO1 in the cell, therebyinhibiting expression of the HAO1 in the cell.

Contacting of a cell with a polynucleotide agent may be done in vitro orin vivo. Contacting a cell in vivo with the polynucleotide agentincludes contacting a cell or group of cells within a subject, e.g., ahuman subject, with the polynucleotide agent. Combinations of in vitroand in vivo methods of contacting are also possible. Contacting may bedirect or indirect, as discussed above. Furthermore, contacting a cellmay be accomplished via a targeting ligand, including any liganddescribed herein or known in the art. In preferred embodiments, thetargeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand, orany other ligand that directs the polynucleotide agent to a site ofinterest, e.g., the liver or pancreas of a subject.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating” and other similar terms, andincludes any level of inhibition.

The phrase “inhibiting expression of an HAO1” is intended to refer toinhibition of expression of any HAO1 gene (such as, e.g., a mouse HAO1gene, a rat HAO1 gene, a monkey HAO1 gene, or a human HAO1 gene) as wellas variants or mutants of an HAO1 gene. Thus, the HAO1 gene may be awild-type HAO1 gene, a mutant HAO1 gene, or a transgenic HAO1 gene inthe context of a genetically manipulated cell, group of cells, ororganism.

“Inhibiting expression of an HAO1 gene” includes any level of inhibitionof an HAO1 gene, e.g., at least partial suppression of the expression ofan HAO1 gene. The expression of the HAO1 gene may be assessed based onthe level, or the change in the level, of any variable associated withHAO1 gene expression, e.g., HAO1 mRNA level, HAO1 protein level, or forexample, oxalate levels can be measured in urine of a subject sufferingfrom PH1 to assess HAO1 expression. The level of HAO1 may be assessed inan individual cell or in a group of cells, including, for example, asample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with HAO1 expressioncompared with a control level. The control level may be any type ofcontrol level that is utilized in the art, e.g., a pre-dose baselinelevel, or a level determined from a similar subject, cell, or samplethat is untreated or treated with a control (such as, e.g., buffer onlycontrol or inactive agent control).

In some embodiments of the methods of the invention, expression of anHAO1 gene is inhibited by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%. at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%. Preferably expression is inhibited by at least about 20%.

Inhibition of the expression of an HAO1 gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which an HAO1 gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with apolynucleotide agent of the invention, or by administering apolynucleotide agent of the invention to a subject in which the cellsare or were present) such that the expression of an HAO1 gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has not or havenot been so treated (control cell(s)). In preferred embodiments, theinhibition is assessed by expressing the level of mRNA in treated cellsas a percentage of the level of mRNA in control cells, using thefollowing formula:

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{nRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, inhibition of the expression of an HAO1 gene may beassessed in terms of a reduction of a parameter that is functionallylinked to HAO1 gene expression, e.g., HAO1 protein expression, mRNA orprotein levels of HAO1 in tissues or serum, e.g., oxalate levels. HAO1gene silencing may be determined in any cell expressing HAO1, eitherconstitutively or by genomic engineering, and by any assay known in theart. The liver is the major site of HAO1 expression. HAO1 is alsoexpressed in the pancreas.

Inhibition of the expression of an HAO1 protein may be manifested by areduction in the level of the HAO1 protein that is expressed by a cellor group of cells (e.g., the level of protein expressed in a samplederived from a subject). As explained above for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of an HAO1 gene includes a cell or group ofcells that has not yet been contacted with a polynucleotide agent of theinvention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with a polynucleotide agent.

The level of HAO1 mRNA that is expressed by a cell or group of cells maybe determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of HAO1 in asample is determined by detecting a transcribed polynucleotide, orportion thereof, e.g., mRNA of the HAO1 gene. RNA may be extracted fromcells using RNA extraction techniques including, for example, using acidphenol/guanidine isothiocyanate extraction (RNAzol® B; Biogenesis),RNeasy RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix®,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays (Melton et al., Nuc. Acids Res. 12:7035), northern blotting, insitu hybridization, and microarray analysis.

In one embodiment, the level of expression of HAO1 is determined using anucleic acid probe. The term “probe”, as used herein, refers to anymolecule that is capable of selectively binding to a specific HAO1.Probes can be synthesized by one of skill in the art, or derived fromappropriate biological preparations. Probes may be specifically designedto be labeled. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to HAO1mRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix® gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of HAO1 mRNA.

An alternative method for determining the level of expression of HAO1 ina sample involves the process of nucleic acid amplification and/orreverse transcriptase (to prepare cDNA) of for example mRNA in thesample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of HAO1 isdetermined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System).

The expression levels of HAO1 mRNA may be monitored using a membraneblot (such as used in hybridization analysis such as northern, Southern,dot, and the like), or microwells, sample tubes, gels, beads or fibers(or any solid support comprising bound nucleic acids). See U.S. Pat.Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of HAO1 expressionlevel may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR).

The level of HAO1 protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, lymph, urine,cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to urine drawn from the subject. In further embodiments,a “sample derived from a subject” refers to liver tissue derived fromthe subject.

In some embodiments of the methods of the invention, the polynucleotideagent is administered to a subject such that the polynucleotide agent isdelivered to a specific site within the subject. The inhibition ofexpression of HAO1 may be assessed using measurements of the level orchange in the level of HAO1 mRNA or HAO1 protein in a sample derivedfrom fluid or tissue from the specific site within the subject. Inpreferred embodiments, the site is the liver. The site may also be asubsection or subgroup of cells from any one of the aforementionedsites. The site may also include cells that express a particular type ofreceptor.

The phrase “contacting a cell with a polynucleotide agent,” as usedherein, includes contacting a cell by any possible means. Contacting acell with a polynucleotide agent includes contacting a cell in vitrowith the polynucleotide agent or contacting a cell in vivo with thepolynucleotide agent. The contacting may be done directly or indirectly.Thus, for example, the polynucleotide agent may be put into physicalcontact with the cell by the individual performing the method, oralternatively, the polynucleotide agent may be put into a situation thatwill permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the polynucleotide agent. Contacting a cell in vivo may bedone, for example, by injecting the polynucleotide agent into or nearthe tissue where the cell is located, or by injecting the polynucleotideagent into another area, e.g., the bloodstream or the subcutaneousspace, such that the agent will subsequently reach the tissue where thecell to be contacted is located. For example, the polynucleotide agentmay contain and/or be coupled to a ligand, e.g., GalNAc3, that directsthe polynucleotide agent to a site of interest, e.g., the liver.Combinations of in vitro and in vivo methods of contacting are alsopossible. For example, a cell may also be contacted in vitro with apolynucleotide agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with a polynucleotide agentincludes “introducing” or “delivering the polynucleotide agent into thecell” by facilitating or effecting uptake or absorption into the cell.Absorption or uptake of a polynucleotide agent can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. Introducing a polynucleotide agent into a cell may be in vitroand/or in vivo. For example, for in vivo introduction, polynucleotideagent can be injected into a tissue site or administered systemically.In vivo delivery can also be done by a beta-glucan delivery system, suchas those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Publication No. 2005/0281781, the entire contents of which are herebyincorporated herein by reference. In vitro introduction into a cellincludes methods known in the art such as electroporation andlipofection. Further approaches are described herein below and/or areknown in the art.

VIII. Methods for Treating or Preventing an HAO1-Associated Disorders

The present invention also provides therapeutic and prophylactic methodswhich include administering to a subject having an HAO1-associateddisease, e.g., primary or secondary hyperoxaluria, a polynucleotideagent or a pharmaceutical composition comprising a polynucleotide agentof the invention. In some aspects of the invention, the methods furtherinclude administering to the subject an additional therapeutic agent,such as vitamin B6 (pyridoxine) and/or potassium citrate, or acombination of any of the foregoing.

In one aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in HAO1expression, e.g., an HAO1-associated disease, e.g., primary or secondaryhyperoxaluria. The treatment methods (and uses) of the invention includeadministering to the subject, e.g., a human, a therapeutically effectiveamount of a polynucleotide agent targeting an HAO1 gene or apharmaceutical composition comprising a polynucleotide agent targetingan HAO1 gene, thereby treating the subject having a disorder that wouldbenefit from reduction in HAO1 expression.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in HAO1expression, e.g., an HAO1-associated disease, e.g., primary or secondaryhyperoxaluria, which include administering to the subject, e.g., ahuman, a therapeutically effective amount of a polynucleotide agenttargeting a HAO1 gene or a pharmaceutical composition comprising apolynucleotide agent targeting an HAO1 gene, and an additionaltherapeutic agent, such as vitamin B6 (pyridoxine) and/or potassiumcitrate, or a combination of any of the foregoing, thereby treating thesubject having a disorder that would benefit from reduction in HAO1expression.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin HAO1 expression, e.g., an HAO1-associated disease, e.g., primary orsecondary hyperoxaluria. The methods include administering to thesubject a prophylactically effective amount of a polynucleotide agenttargeting an HAO1 gene or a pharmaceutical composition comprising apolynucleotide agent targeting an HAO1 gene, thereby preventing at leastone symptom in the subject having a disorder that would benefit fromreduction in HAO1 expression. For example, the invention providesmethods for reducing or preventing oxalate deposition in a subjectsuffering from a disorder that would benefit from reduction in HAO1expression.

In certain aspects, the invention provides methods of preventing atleast one symptom in a subject having a disorder that would benefit fromreduction in HAO1 expression, e.g., an HAO1-associated disease, e.g.,primary or secondary hyperoxaluria. The methods include administering tothe subject a prohpylactically effective amount of a polynucleotideagent targeting an HAO1 gene or a pharmaceutical composition comprisinga polynucleotide agent targeting an HAO1 gene, and an additionaltherapeutic agent, such as vitamin B6 (pyridoxine) and/or potassiumcitrate, thereby preventing at least one symptom in the subject having adisorder that would benefit from reduction in HAO1 expression.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of a polynucleotide agent or another agent fortreatment of an HAO1-associated disease, e.g., vitamin B6 (pyridoxine)and/or potassium citrate, or a combination of any of the foregoing,that, when administered to a subject having an HAO1-associated disease,is sufficient to effect treatment of the disease (e.g., by diminishing,ameliorating or maintaining the existing disease or one or more symptomsof disease). The “therapeutically effective amount” may vary dependingon the polynucleotide agent or the other agent(s) for treatment of theHAO1-associated disease, how the agent is administered, the disease andits severity and the history, age, weight, family history, geneticmakeup, the types of preceding or concomitant treatments, if any, andother individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of a polynucleotide agent other agent(s) fortreatment of the HAO1-associated disease, that, when administered to asubject having an HAO1-associated disease but not yet (or currently)experiencing or displaying symptoms of the disease, and/or a subject atrisk of developing an HAO1-associated disease, e.g., a subject whocarries a mutation in the AGXT gene, is sufficient to prevent orameliorate the disease or one or more symptoms of the disease.Ameliorating the disease includes slowing the course of the disease orreducing the severity of later-developing disease. The “prophylacticallyeffective amount” may vary depending on the polynucleotide agent oragent(s) for treatment of the HAO1-associated disease, how the agent(s)is administered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically effective amount” or “prophylactically effectiveamount” also includes an amount of a polynucleotide agent or otheragent(s) for treatment of the HAO1-associated disease, that producessome desired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. Polynucleotide agents employed in themethods of the present invention may be administered in a sufficientamount to produce a reasonable benefit/risk ratio applicable to suchtreatment.

In another aspect, the present invention provides uses of atherapeutically effective amount of a polynucleotide agent of theinvention for treating a subject, e.g., a subject that would benefitfrom a reduction and/or inhibition of HAO1 expression.

In another aspect, the present invention provides uses of atherapeutically effective amount of a polynucleotide agent of theinvention and an additional therapeutic agent(s) for treatment of theHAO1-associated disease for treating a subject, e.g., a subject thatwould benefit from a reduction and/or inhibition of HAO1 expression.

In yet another aspect, the present invention provides use of apolynucleotide agent of the invention targeting an HAO1 gene or apharmaceutical composition comprising a polynucleotide agent targetingan HAO1 gene in the manufacture of a medicament for treating a subject,e.g., a subject that would benefit from a reduction and/or inhibition ofHAO1 expression, such as a subject having a disorder that would benefitfrom reduction in HAO1 expression, e.g., an HAO1-associated disease,e.g., primary or secondary hyperoxaluria.

In another aspect, the present invention provides uses of apolynucleotide agent of the invention targeting an HAO1 gene or apharmaceutical composition comprising a polynucleotide agent targetingan HAO1 gene in the manufacture of a medicament for use in combinationwith an additional therapeutic agent for treatment of theHAO1-associated diseases, such as vitamin B6 (pyridoxine) and/orpotassium citrate, or a combination of any of the foregoing, fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of HAO1 expression, e.g., an HAO1-associated disease,e.g., primary or secondary hyperoxaluria.

In another aspect, the invention provides uses of a polynucleotide agentof the invention for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of HAO1 expression, such as an HAO1-associated disease, e.g.,primary or secondary hyperoxaluria.

In yet another aspect, the invention provides uses of a polynucleotideagent of the invention, and an additional therapeutic agent fortreatment of the HAO1-associated diseases, such as vitamin B6(pyridoxine) and/or potassium citrate, or a combination of any of theforegoing, for preventing at least one symptom in a subject sufferingfrom a disorder that would benefit from a reduction and/or inhibition ofHAO1, such as and HAO1-associated disease, e.g., primary or secondaryhyperoxaluria.

In a further aspect, the present invention provides uses of apolynucleotide agent of the invention in the manufacture of a medicamentfor preventing at least one symptom in a subject suffering from adisorder that would benefit from a reduction and/or inhibition of HAO1expression, such as an HAO1-associated disease, e.g., primary orsecondary hyperoxaluria.

In a further aspect, the present invention provides uses of apolynucleotide agent of the invention in the manufacture of a medicamentfor use in combination with an additional therapeutic agent, such asvitamin B6 (pyridoxine) and/or potassium citrate, or a combination ofany of the foregoing, for preventing at least one symptom in a subjectsuffering from a disorder that would benefit from a reduction and/orinhibition of HAO1 expression, such as an HAO1-associated disease, e.g.,primary or secondary hyperoxaluria.

In one embodiment, a polynucleotide agent targeting HAO1 is administeredto a subject having an HAO1-associated disease such that HAO1 levels,e.g., in a cell, tissue, blood, urine or other tissue or fluid of thesubject are reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% or more and, subsequently, an additional therapeutic (asdescribed below) is administered to the subject.

The additional therapeutic agent for the treatment of an HAO1-associateddisease may be, for example, vitamin B6 (pyridoxine) and/or potassiumcitrate.

In exemplary methods of the invention for treating an HAO1-associateddisease, e.g., primary or secondary hyperoxaluria, a polynucleotideagent targeting HAO1 is administered (e.g., subcutaneously) to thesubject first, such that the HAO1 levels in the subject are reduced(e.g., by at least about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, orat least about 99% or more) and subsequently for treatment of theHAO1-associated diseases is administered at doses lower than the onesdescribed in the product insert for the agent. In certain embodiments,the agent for treatment of the HAO1-associated disease can beadministered at a sufficiently low dose to decrease side effects to anacceptable level.

The methods and uses of the invention include administering acomposition described herein such that expression of the target HAO1gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18,24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80hours. In certain embodiments, expression of the target HAO1 gene isdecreased for an extended duration, e.g., at least about two, three,four, five, six, seven days or more, e.g., about one week, two weeks,three weeks, or about four weeks or longer.

Administration of the polynucleotide agent according to the methods anduses of the invention may result in a reduction of the severity, signs,symptoms, and/or markers of such diseases or disorders in a patient withan HAO1-associated disease. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of an HAO1-associated disorder, e.g.,primary or secondary hyperoxaluria, may be assessed, for example, byperiodic monitoring of oxalate levels in the subject being treated.Comparisons of the later measurements with the initial measurementsprovide a physician an indication of whether the treatment is effective.It is well within the ability of one skilled in the art to monitorefficacy of treatment or prevention by measuring such a parameter, orany combination of parameters. In connection with the administration ofa polynucleotide agent targeting HAO1 or pharmaceutical compositionthereof, “effective against” an HAO1-associated disease indicates thatadministration in a clinically appropriate manner results in abeneficial effect for at least a statistically significant fraction ofpatients, such as improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating an HAO1-associated disease and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given polynucleotide agent drug or formulationof that drug can also be judged using an experimental animal model forthe given disease as known in the art. When using an experimental animalmodel, efficacy of treatment is evidenced when a statisticallysignificant reduction in a marker or symptom is observed.

Any positive change resulting in e.g., lessening of severity of diseasemeasured using the appropriate scale, represents adequate treatmentusing a polynucleotide agent or polynucleotide agent formulation asdescribed herein.

Subjects can be administered a therapeutic amount of polynucleotideagent, such as about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg,0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg,0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg,0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg,0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg,2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg,3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg, 4.1mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg,4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3 mg/kg, 5.4mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg, 6.0 mg/kg,6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg, 6.7mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3 mg/kg,7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9 mg/kg, 8.0mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg, 8.6 mg/kg,8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2 mg/kg, 9.3mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg,9.0 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg,40 mg/kg, 45 mg/kg, or about 50 mg/kg. Values and ranges intermediate tothe recited values are also intended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a polynucleotide agent as described herein and a lipid,subjects can be administered a therapeutic amount of polynucleotideagent, such as about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg toabout 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg toabout 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg toabout 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg toabout 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg toabout 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg toabout 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg toabout 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg,about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5mg/kg to about 10 mg/kg. Values and ranges intermediate to the recitedvalues are also intended to be part of this invention.

For example, the polynucleotide agent may be administered at a dose ofabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

In other embodiments, subjects can be administered a therapeutic amountof polynucleotide agent, such as a dose of about 0.1 to about 50 mg/kg,about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 toabout 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb,about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20mg/kg. In one embodiment, when a composition of the invention comprisesa polynucleotide agent as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of about 10 to about30 mg/kg of polynucleotide agent. Values and ranges intermediate to therecited values are also intended to be part of this invention.

For example, subjects can be administered a therapeutic amount ofpolynucleotide agent, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5,16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5,23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5,30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to therecited values are also intended to be part of this invention.

The polynucleotide agent can be administered by intravenous infusionover a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minute period. Theadministration may be repeated, for example, on a regular basis, such asweekly, biweekly (i.e., every two weeks) for one month, two months,three months, four months or longer. After an initial treatment regimen,the treatments can be administered on a less frequent basis. Forexample, after administration weekly or biweekly for three months,administration can be repeated once per month, for six months or a yearor longer.

Administration of the polynucleotide agent can reduce HAO1 levels, e.g.,in a cell, tissue, blood, urine or other compartment of the patient byat least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% or more.

Before administration of a full dose of the polynucleotide agent,patients can be administered a smaller dose, such as a 5% infusion, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Owing to the inhibitory effects on HAO1 expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

A polynucleotide agent of the invention may be administered in “naked”form, or as a “free polynucleotide agent.” A naked polynucleotide agentis administered in the absence of a pharmaceutical composition. Thenaked polynucleotide agent may be in a suitable buffer solution. Thebuffer solution may comprise acetate, citrate, prolamine, carbonate, orphosphate, or any combination thereof. In one embodiment, the buffersolution is phosphate buffered saline (PBS). The pH and osmolarity ofthe buffer solution containing the polynucleotide agent can be adjustedsuch that it is suitable for administering to a subject.

Alternatively, a polynucleotide agent of the invention may beadministered as a pharmaceutical composition, such as a polynucleotideagent liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of HAO1gene expression are those having an HAO1-associated disease or disorderas described herein. In certain embodiments, a subject having anHAO1-associated disease has primary or secondary hyperoxaluria.

Treatment of a subject that would benefit from a reduction and/orinhibition of HAO1 gene expression includes therapeutic and prophylactictreatment (e.g., the subject carries a mutation in the AGTX gene and hasPH1).

The invention further provides methods and uses of a polynucleotideagent or a pharmaceutical composition thereof (including methods anduses of a polynucleotide agent or a pharmaceutical compositioncomprising a polynucleotide agent and an for treatment of theHAO1-associated diseases) for treating a subject that would benefit fromreduction and/or inhibition of HAO1 expression, e.g., a subject havingan HAO1-associated disease, in combination with other pharmaceuticalsand/or other therapeutic methods, e.g., with known pharmaceuticalsand/or known therapeutic methods, such as, for example, those which arecurrently employed for treating these disorders. For example, in certainembodiments, a polynucleotide agent targeting HAO1 is administered incombination with, e.g., an agent useful in treating an HAO1-associateddisease as described elsewhere herein.

For example, additional therapeutics and therapeutic methods suitablefor treating a subject that would benefit from reduction in HAO1expression, e.g., a subject having an HAO1-associated disease, includevitamin B6 (pyridoxine) and/or potassium citrate, or a combination ofany of the foregoing.

The polynucleotide agent (and/or agent(s) for treatment of theHAO1-associated disease) and an additional therapeutic agent and/ortreatment may be administered at the same time and/or in the samecombination, e.g., parenterally, or the additional therapeutic agent canbe administered as part of a separate composition or at separate timesand/or by another method known in the art or described herein.

The present invention also provides methods of using a polynucleotideagent, e.g., a antisense polynucleotide agent, of the invention and/or acomposition containing a polynucleotide agent of the invention to reduceand/or inhibit HAO1 expression in a cell. In other aspects, the presentinvention provides a polynucleotide agent of the invention and/or acomposition comprising a polynucleotide agent of the invention for usein reducing and/or inhibiting HAO1 expression in a cell. In yet otheraspects, use of a polynucleotide agent of the invention and/or acomposition comprising a polynucleotide agent of the invention for themanufacture of a medicament for reducing and/or inhibiting HAO1expression in a cell are provided.

The methods and uses include contacting the cell with a polynucleotideagent, e.g., a antisense polynucleotide agent, of the invention andmaintaining the cell for a time sufficient to obtain antisenseinhibition of a HAO1 gene, thereby inhibiting expression of the HAO1gene in the cell.

Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of HAO1 may bedetermined by determining the mRNA expression level of HAO1 usingmethods routine to one of ordinary skill in the art, e.g., northernblotting, qRT-PCR, by determining the protein level of HAO1 usingmethods routine to one of ordinary skill in the art, such as westernblotting, immunological techniques, flow cytometry methods, ELISA,and/or by determining a biological activity of HAO1, such as monitoringof oxalate levels.

In the methods and uses of the invention the cell may be contacted invitro or in vivo, i.e., the cell may be within a subject. In embodimentsof the invention in which the cell is within a subject, the methods mayinclude further contacting the cell with an agent for treatment of theHAO1-associated disease.

A cell suitable for treatment using the methods of the invention may beany cell that expresses an HAO1 gene. A cell suitable for use in themethods and uses of the invention may be a mammalian cell, e.g., aprimate cell (such as a human cell or a non-human primate cell, e.g., amonkey cell or a chimpanzee cell), a non-primate cell (such as a cowcell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell,a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, adog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bearcell, or a buffalo cell), a bird cell (e.g., a duck cell or a goosecell), or a whale cell. In one embodiment, the cell is a human cell,e.g., a human liver cell.

HAO1 expression may be inhibited in the cell by at least about 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administeringto a subject a composition containing a polynucleotide agent, where thepolynucleotide agent includes a nucleotide sequence that iscomplementary to at least a part of an RNA transcript of the HAO1 geneof the mammal to be treated. When the organism to be treated is a mammalsuch as a human, the composition can be administered by any means knownin the art including, but not limited to subcutaneous, intravenous,oral, intraperitoneal, or parenteral routes, including intracranial(e.g., intraventricular, intraparenchymal and intrathecal),intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the polynucleotide agent in a consistent wayover a prolonged time period. Thus, a depot injection may reduce thefrequency of dosing needed to obtain a desired effect, e.g., a desiredinhibition of HAO1, or a therapeutic or prophylactic effect. A depotinjection may also provide more consistent serum concentrations. Depotinjections may include subcutaneous injections or intramuscularinjections. In preferred embodiments, the depot injection is asubcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the polynucleotide agent to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of an HAO1 gene in a mammal, e.g., a human.The present invention also provides a composition comprising apolynucleotide agent that targets an HAO1 gene in a cell of a mammal foruse in inhibiting expression of the HAO1 gene in the mammal. In anotheraspect, the present invention provides use of a polynucleotide agentthat targets an HAO1 gene in a cell of a mammal in the manufacture of amedicament for inhibiting expression of the HAO1 gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human,a composition comprising a polynucleotide agent that targets an HAO1gene in a cell of the mammal and maintaining the mammal for a timesufficient to obtain antisense inhibition of the mRNA transcript of theHAO1 gene, thereby inhibiting expression of the HAO1 gene in the mammal.In some embodiment, the methods further comprise administering an agentfor treatment of the HAO1-associated disease to the subject.

Reduction in gene expression can be assessed by any methods known it theart and by methods, e.g. qRT-PCR, described herein. Reduction in proteinproduction can be assessed by any methods known it the art and bymethods, e.g., ELISA or western blotting, described herein. In oneembodiment, a puncture liver biopsy sample serves as the tissue materialfor monitoring the reduction in HAO1 gene and/or protein expression. Inanother embodiment, a blood sample serves as the tissue material formonitoring the reduction in HAO1 gene and/or protein expression.Suitable assays are further described in the Examples section below.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the polynucleotide agents and methods featured inthe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

EXAMPLES Example 1. Design and Synthesis of Antisense Polynucleotides

The antisense polynucleotides targeting HAO1 were synthesized usingstandard synthesis methods well known in the art.

A detailed list of antisense molecules targeting HAO1 is shown in Table3 for modified antisense molecules and in Table 4 for unmodifiedantisense molecules.

Example 2. In Vitro Screening

In vitro screening of the antisense polynucleotides was performed bytransfecting primary human hepatocytes with 500 nM of each singlestranded antisense oligonucleotide (ASO). Specifically, 0.4 μl ofRNAiMax (Invitrogen) was incubated with each ASO according to themanufacturer's protocol and then added to 3.2×10⁴ cells in each well ofa 96 well plate. Plates were incubated for 24 hours prior to harvestingfor analysis.

For measurement of HAO1 mRNA, cells were harvested 24 h aftertransfection and lysed at 53° C. following procedures recommended by themanufacturer of the Quantigene II Kit for HAO1 and Quantigene I ExploreKit for (Panomics, Fremont, Calif., USA) bDNA. Afterwards, 50 μl of eachlysate was incubated with probe sets specific to human HAO1 and 10 μl ofeach lysate was incubated with the probe set specific for human GAPDH.After incubation, each lysate was processed according to themanufacturer's protocol for QuantiGene. Chemoluminescence was measuredin a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relativelight units) and values obtained with the human HAO1 probeset werenormalized to the respective human GAPDH values for each well and thenrelated to the mean of an unrelated control gene. Table 5 shows theresults of single dose transfection screen in cells transfected with theindicated antisense polynucleotide.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3'-phosphate Af 2'-fluoroadenosine-3'-phosphate Afs2'-fluoroadenosine-3'-phosphorothioate As adenosine-3'-phosphorothioatea 2′-O-methyladenosine-3'-phosphate as2′-O-methyladenosine-3'-phosphorothioate C cytidine-3'-phosphate dA2{grave over ( )}-deoxyadenosine-3{grave over ( )}-phosphate dAs 2{graveover ( )}-deoxyadenosine-3{grave over ( )}-phosphorothioate Cf2'-fluorocytidine-3'-phosphate Cfs 2'-fluorocytidine-3'-phosphorothioateCs cytidine-3'-phosphorothioate c 2′-O-methylcytidine-3'-phosphate cs2′-O-methylcytidine-3'-phosphorothioate dC 2{grave over( )}-deoxycytidine-3{grave over ( )}-phosphate dCs 2{grave over( )}-deoxycytidine-3{grave over ( )}-phosphorothioate Gguanosine-3'-phosphate Gf 2'-fluoroguanosine-3'-phosphate Gfs2'-fluoroguanosine-3'-phosphorothioate Gs guanosine-3'-phosphorothioateg 2′-O-methylguanosine-3'-phosphate gs2′-O-methylguanosine-3'-phosphorothioate dG 2{grave over( )}-deoxyguanosine-3{grave over ( )}-phosphate dGs 2{grave over( )}-deoxyguanosine-3{grave over ( )}-phosphorothioate T5'-methyluridine-3'-phosphate Tf 2'-fluoro-5-methyluridine-3'-phosphateTfs 2'-fluoro-5-methyluridine-3'-phosphorothioate Ts5-methyluridine-3'-phosphorothioate t2'-O-methyl-5-methyluridine-3'-phosphate ts2'-O-methyl-5-methyluridine-3'-phosphorothioate dT 2{grave over( )}-deoxythymidine-3{grave over ( )}-phosphate dTs 2{grave over( )}-deoxythymidine-3{grave over ( )}-phosphorothioate UUridine-3'-phosphate Uf 2'-fluorouridine-3'-phosphate Ufs2'-fluorouridine-3'-phosphorothioate Us uridine-3'-phosphorothioate u2′-O-methyluridine-3'-phosphate us2′-O-methyluridine-3'-phosphorothioate dU 2{grave over( )}-deoxyuridine-3'-phosphate dUs 2{grave over( )}-deoxyuridine-3{grave over ( )}-phosphorothioate s phosphorothioatelinkage N any nucleotide (G, A, C, T or U) L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3 (dt) deoxy-thymine (5MdC)5'-methyl-deoxycytidine-3{grave over ( )}-phosphate (5MdC)s5'-methyl-deoxycytidine-3{grave over ( )}-phosphorothioate

TABLE 3 Modified antisense polynucleotides targeting HAO1. SEQ ID TargetOligo Name Sequence 5′-3′ NO: HAO1 A-133284.1gsgsgsasgs(5MdC)sdAsdTsdTsdTsdTs(5MdC)sdAs(5MdC)sdAsgsgsususa  9 HAO1A-133285.1 asasususasdGs(5MdC)s(5MdC)sdGsdGsdGsdGsdGsdAsdGscsasususu 10HAO1 A-133286.1 asuscsasusdTsdGsdAsdTsdAs(5MdC)sdAsdAsdAsdTsusasgscsc 11HAO1 A-133287.1gsususgsusdTs(5MdC)sdAsdTsdAsdAsdTs(5MdC)sdAsdTsusgsasusa 12 HAO1A-133288.1 gsasusususdAsdGs(5MdC)sdAsdTsdGsdTsdTsdGsdTsuscsasusa 13 HAO1A-133289.1 ususgsgsasdAsdGsdTsdAs(5MdC)sdTsdGsdAsdTsdTsusasgscsa 14 HAO1A-133290.1 csasusasusdAsdTsdAsdGsdAs(5MdC)sdTsdTsdTsdGsgsasasgsu 15 HAO1A-133291.1 csusgsusasdAsdTsdAsdGsdTs(5MdC)sdAsdTsdAsdTsasusasgsa 16 HAO1A-133292.1ususgscscs(5MdC)s(5MdC)sdAsdGsdAs(5MdC)s(5MdC)sdTsdGsdTsasasusasg 17HAO1 A-133293.1ususcsusus(5MdC)sdAsdTs(5MdC)sdAsdTsdTsdTsdGs(5MdC)scscscsasg 18 HAO1A-133294.1 usasuscsasdGs(5MdC)s(5MdC)sdAsdAsdAsdGsdTsdTsdTscsususcsa 19HAO1 A-133295.1 gscsusgscsdAsdAsdTsdAsdTsdTsdAsdTs(5MdC)sdAsgscscsasa 20HAO1 A-133296.1 asuscsusgsdGsdAsdAsdAsdAsdTsdGs(5MdC)sdTsdGscsasasusa 21HAO1 A-133297.1gsasusascsdAsdGs(5MdC)sdTsdTs(5MdC)s(5MdC)sdAsdTs(5MdC)susgsgsasa 22HAO1 A-133298.1gsasgscsasdTs(5MdC)s(5MdC)sdTsdTsdGsdGsdAsdTsdAscsasgscsu 23 HAO1A-133299.1 csasascsasdTsdTs(5MdC)s(5MdC)sdGsdGsdAsdGs(5MdC)sdAsuscscsusu24 HAO1 A-133300.1gsasuscsusdGsdTsdTsdTs(5MdC)sdAsdGs(5MdC)sdAsdAscsasususc 25 HAO1A-133301.1 asgsasasgsdTs(5MdC)sdGsdAs(5MdC)sdAsdGsdAsdTs(5MdC)susgsususu26 HAO1 A-133302.1 usgsuscscsdTsdAsdAsdAsdAs(5MdC)sdAsdGsdAsdAsgsuscsgsa27 HAO1 A-133303.1usgscsusgsdAs(5MdC)s(5MdC)s(5MdC)sdTs(5MdC)sdTsdGsdTs(5MdC)scsusasasa 28HAO1 A-133304.1csasusasusdTsdGsdGs(5MdC)sdAsdTsdGs(5MdC)sdTsdGsascscscsu 29 HAO1A-133305.1asgscscscs(5MdC)s(5MdC)sdAs(5MdC)sdAs(5MdC)sdAsdTsdAsdTsusgsgscsa 30HAO1 A-133306.1csusgscsasdTsdGsdGs(5MdC)s(5MdC)sdGsdTsdAsdGs(5MdC)scscscscsa 31 HAO1A-133307.1 gsasgscscsdAsdTsdGs(5MdC)sdGs(5MdC)sdTsdGs(5MdC)sdAsusgsgscsc32 HAO1 A-133308.1gscscsgsus(5MdC)s(5MdC)sdAs(5MdC)sdAsdTsdGsdAsdGs(5MdC)scsasusgsc 33HAO1 A-133309.1asgsusgsgs(5MdC)sdAsdAsdGs(5MdC)sdTs(5MdC)sdGs(5MdC)s(5MdC)sgsuscscsa 34HAO1 A-133310.1ascsasgsgs(5MdC)sdTs(5MdC)sdTs(5MdC)sdAs(5MdC)sdAsdGsdTsgsgscsasa 35HAO1 A-133311.1csasgsgsgsdAs(5MdC)sdTsdGsdAs(5MdC)sdAsdGsdGs(5MdC)suscsuscsa 36 HAO1A-133312.1asusgscscs(5MdC)sdGsdTsdTs(5MdC)s(5MdC)s(5MdC)sdAsdGsdGsgsascsusg 37HAO1 A-133313.1gsasascsus(5MdC)sdAsdAs(5MdC)sdAsdTs(5MdC)sdAsdTsdGscscscsgsu 38 HAO1A-133314.1 gsasgsgsusdGsdGs(5MdC)s(5MdC)s(5MdC)sdAsdGsdGsdAsdAscsuscsasa39 HAO1 A-133315.1 uscsasasusdTsdGsdAsdGsdGsdAsdGsdGsdTsdGsgscscscsa 40HAO1 A-133316.1cscsgscscsdAs(5MdC)sdTsdTs(5MdC)sdTsdTs(5MdC)sdAsdAsususgsasg 41 HAO1A-133317.1csasgsgsas(5MdC)s(5MdC)sdAsdGs(5MdC)sdTsdTs(5MdC)s(5MdC)sdGscscsascsu 42HAO1 A-133318.1ascsgsasasdGsdTsdGs(5MdC)s(5MdC)sdTs(5MdC)sdAsdGsdGsascscsasg 43 HAO1A-133319.1csasgsususdGs(5MdC)sdAsdGs(5MdC)s(5MdC)sdAsdAs(5MdC)sdGsasasgsusg 44HAO1 A-133320.1 usgsusasgsdAsdTsdAsdTsdAs(5MdC)sdAsdGsdTsdTsgscsasgsc 45HAO1 A-133321.1uscsuscsgsdGsdTs(5MdC)s(5MdC)sdTsdTsdGsdTsdAsdGsasusasusa 46 HAO1A-133322.1 ususcsususdGsdGsdTsdGsdAs(5MdC)sdTsdTs(5MdC)sdTscsgsgsusc 47HAO1 A-133323.1cscsgscsas(5MdC)sdTsdAsdGs(5MdC)sdTsdTs(5MdC)sdTsdTsgsgsusgsa 48 HAO1A-133324.1csususcsus(5MdC)sdTsdGs(5MdC)s(5MdC)sdTsdGs(5MdC)s(5MdC)sdGscsascsusa 49HAO1 A-133325.1csususgsusdAsdGs(5MdC)s(5MdC)s(5MdC)sdAsdTs(5MdC)sdTsdTscsuscsusg 50HAO1 A-133326.1asasasusasdTsdGsdGs(5MdC)s(5MdC)sdTsdTsdGsdTsdAsgscscscsa 51 HAO1A-133327.1 usgsuscscsdAs(5MdC)sdTsdGsdTs(5MdC)sdAs(5MdC)sdAsdAsasusasusg52 HAO1 A-133328.1 csasgsgsusdAsdAsdGsdGsdTsdGsdTsdGsdTs(5MdC)scsascsusg53 HAO1 A-133329.1gsascsgsgsdTsdTsdGs(5MdC)s(5MdC)s(5MdC)sdAsdGsdGsdTsasasgsgsu 54 HAO1A-133330.1csascsasus(5MdC)sdAsdTs(5MdC)s(5MdC)sdAsdGsdAs(5MdC)sdGsgsususgsc 55HAO1 A-133331.1asasuscsusdGsdTsdTsdAs(5MdC)sdGs(5MdC)sdAs(5MdC)sdAsuscsasusc 56 HAO1A-133332.1 gscsgsgscsdAsdGsdTsdTsdTsdGsdAsdAsdTs(5MdC)susgsususa 57 HAO1A-133333.1 csusgsasgsdTsdTsdGsdTsdGsdGs(5MdC)sdGsdGs(5MdC)sasgsususu 58HAO1 A-133334.1asasasasusdTsdTsdTsdTs(5MdC)sdAsdTs(5MdC)s(5MdC)sdTsgsasgsusu 59 HAO1A-133335.1 usascsusgsdGsdTsdTsdTs(5MdC)sdAsdAsdAsdAsdTsususususc 60 HAO1A-133336.1 asasasusgsdAsdTsdAsdAsdAsdGsdTsdAs(5MdC)sdTsgsgsususu 61 HAO1A-133337.1 cscsuscsasdGsdGsdAsdGsdAsdAsdAsdAsdTsdGsasusasasa 62 HAO1A-133338.1 uscscsasasdAsdAsdTsdTsdTsdTs(5MdC)s(5MdC)sdTs(5MdC)sasgsgsasg63 HAO1 A-133339.1gsuscscsas(5MdC)sdTsdGsdTs(5MdC)sdGsdTs(5MdC)sdTs(5MdC)scsasasasa 64HAO1 A-133340.1asusasusgs(5MdC)sdAsdGs(5MdC)sdAsdAsdGsdTs(5MdC)s(5MdC)sascsusgsu 65HAO1 A-133341.1usususasgs(5MdC)s(5MdC)sdAs(5MdC)sdAsdTsdAsdTsdGs(5MdC)sasgscsasa 66HAO1 A-133342.1usgsgsgsus(5MdC)sdTsdAsdTsdTsdGs(5MdC)sdTsdTsdTsasgscscsa 67 HAO1A-133343.1 asgscsusgsdAsdTsdAsdGsdAsdTsdGsdGsdGsdTscsusasusu 68 HAO1A-133344.1gsasusasus(5MdC)sdTsdTs(5MdC)s(5MdC)s(5MdC)sdAsdGs(5MdC)sdTsgsasusasg 69HAO1 A-133345.1csuscsasgs(5MdC)s(5MdC)sdAsdTsdTsdTsdGsdAsdTsdAsuscsususc 70 HAO1A-133346.1gsasusgsus(5MdC)sdAsdGsdTs(5MdC)sdTsdTs(5MdC)sdTs(5MdC)sasgscscsa 71HAO1 A-133347.1 csasasususdGsdGs(5MdC)sdAsdAsdTsdGsdAsdTsdGsuscsasgsu 72HAO1 A-133348.1cscscsususdTsdGs(5MdC)sdAsdAs(5MdC)sdAsdAsdTsdTsgsgscsasa 73 HAO1A-133349.1 csuscsuscsdAsdAsdAsdAsdTsdGs(5MdC)s(5MdC)s(5MdC)sdTsususgscsa74 HAO1 A-133350.1gsgscsasus(5MdC)sdAsdTs(5MdC)sdAs(5MdC)s(5MdC)sdTs(5MdC)sdTscsasasasa 75HAO1 A-133351.1asascsasgs(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)s(5MdC)sdTsdGsdGs(5MdC)sasuscsasu76 HAO1 A-133352.1 asasgscscsdAsdTsdGsdTsdTsdTsdAsdAs(5MdC)sdAsgscscsusc77 HAO1 A-133353.1asasgsasus(5MdC)s(5MdC)s(5MdC)sdAsdTsdTs(5MdC)sdAsdAsdGscscsasusg 78HAO1 A-133354.1ususcsgsas(5MdC)sdAs(5MdC)s(5MdC)sdAsdAsdGsdAsdTs(5MdC)scscsasusu 79HAO1 A-133355.1uscsgsasgs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdAsdTsdGsdAsdTsdTscsgsascsa 80HAO1 A-133356.1asuscsgsasdGsdTsdTsdGsdTs(5MdC)sdGsdAsdGs(5MdC)scscscsasu 81 HAO1A-133357.1gsgscsusgsdGs(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdAsdTs(5MdC)sgsasgsusu82 HAO1 A-133358.1asascsasus(5MdC)sdAsdAsdTsdAsdGsdTsdGsdGs(5MdC)susgsgscsa 83 HAO1A-133359.1 asusususcsdTsdGsdGs(5MdC)sdAsdGsdAsdAs(5MdC)sdAsuscsasasu 84HAO1 A-133360.1asgscscsus(5MdC)s(5MdC)sdAs(5MdC)sdAsdAsdTsdTsdTs(5MdC)susgsgscsa 85HAO1 A-133361.1csususcscs(5MdC)sdTsdTs(5MdC)s(5MdC)sdAs(5MdC)sdAsdGs(5MdC)scsuscscsa 86HAO1 A-133362.1asasgsascsdTsdTs(5MdC)s(5MdC)sdAs(5MdC)s(5MdC)sdTsdTs(5MdC)scscsususc 87HAO1 A-133363.1cscsgsuscs(5MdC)sdAsdGsdGsdAsdAsdGsdAs(5MdC)sdTsuscscsasc 88 HAO1A-133364.1uscscsgscsdAs(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)s(5MdC)s(5MdC)sdGsdTscscsasgsg89 HAO1 A-133365.1csasuscsasdGsdTsdGs(5MdC)s(5MdC)sdTsdTsdTs(5MdC)s(5MdC)sgscsascsa 90HAO1 A-133366.1asgscsususdTs(5MdC)sdAsdGsdAsdAs(5MdC)sdAsdTs(5MdC)sasgsusgsc 91 HAO1A-133367.1 csasasgsasdGs(5MdC)s(5MdC)sdAsdGsdAsdGs(5MdC)sdTsdTsuscsasgsa92 HAO1 A-133368.1ascsasgscs(5MdC)sdTsdTsdGsdGs(5MdC)sdGs(5MdC)s(5MdC)sdAsasgsasgsc 93HAO1 A-133369.1uscscscscsdAs(5MdC)sdAsdAsdAs(5MdC)sdAs(5MdC)sdAsdGscscsususg 94 HAO1A-133370.1ascsgsasusdTsdGsdGsdTs(5MdC)sdTs(5MdC)s(5MdC)s(5MdC)s(5MdC)sascsasasa 95HAO1 A-133371.1usasasgscs(5MdC)s(5MdC)s(5MdC)sdAsdAsdAs(5MdC)sdGsdAsdTsusgsgsusc 96HAO1 A-133372.1 cscscsusgsdGsdAsdAsdAsdGs(5MdC)sdTsdAsdAsdGscscscscsa 97HAO1 A-133373.1csascscsusdTsdTs(5MdC)sdTs(5MdC)s(5MdC)s(5MdC)s(5MdC)s(5MdC)sdTsgsgsasasa98 HAO1 A-133374.1gsascsasus(5MdC)sdTsdTsdGsdAsdAs(5MdC)sdAs(5MdC)s(5MdC)susususcsu 99HAO1 A-133375.1usasgsusasdTs(5MdC)sdTs(5MdC)sdGsdAsdGsdGsdAs(5MdC)sasuscsusu 100 HAO1A-133376.1 gsasasusus(5MdC)sdTsdTs(5MdC)s(5MdC)sdTsdTsdTsdAsdGsusasuscsu101 HAO1 A-133377.1gsgscscsasdAs(5MdC)s(5MdC)sdGsdGsdAsdAsdTsdTs(5MdC)sususcscsu 102 HAO1A-133378.1csuscsasgsdAsdGs(5MdC)s(5MdC)sdAsdTsdGsdGs(5MdC)s(5MdC)sasascscsg 103HAO1 A-133379.1asususcsusdGsdGs(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)sdAs(5MdC)sdTscsasgsasg104 HAO1 A-133380.1asusgsascsdTsdTsdTs(5MdC)sdAs(5MdC)sdAsdTsdTs(5MdC)susgsgscsa 105 HAO1A-133381.1 gsuscsususdGsdTs(5MdC)sdGsdAsdTsdGsdAs(5MdC)sdTsususcsasc 106HAO1 A-133382.1usususcscsdTs(5MdC)sdAs(5MdC)s(5MdC)sdAsdAsdTsdGsdTscsususgsu 107 HAO1A-133383.1 csasasasgsdGsdAsdTsdTsdTsdTsdTs(5MdC)s(5MdC)sdTscsascscsa 108HAO1 A-133384.1ususgsgsasdAsdAs(5MdC)sdGsdGs(5MdC)s(5MdC)sdAsdAsdAsgsgsasusu 109 HAO1A-133385.1 gscsascsusdGsdTs(5MdC)sdAsdGsdAsdTs(5MdC)sdTsdTsgsgsasasa 110HAO1 A-133386.1asasasusasdTsdTsdGsdTsdGs(5MdC)sdAs(5MdC)sdTsdGsuscsasgsa 111 HAO1A-133387.1 usascsasgsdAsdTsdGsdGsdGsdAsdAsdAsdAsdTsasususgsu 112 HAO1A-133388.1 usgsasasasdAsdAsdAsdAsdAsdTsdAsdAsdTsdAscsasgsasu 113 HAO1A-133389.1 usasasusas(5MdC)sdAsdTsdGs(5MdC)sdTsdGsdAsdAsdAsasasasasa 114HAO1 A-133390.1 csuscsususdTsdGsdTs(5MdC)sdAsdAsdGsdTsdAsdAsusascsasu115 HAO1 A-133391.1gscsascsasdGsdTsdGsdTs(5MdC)sdTs(5MdC)sdTsdTsdTsgsuscsasa 116 HAO1A-133392.1usgsgsuscsdAs(5MdC)s(5MdC)s(5MdC)sdTs(5MdC)sdTsdGs(5MdC)sdAscsasgsusg117 HAO1 A-133393.1ususascsasdGsdAs(5MdC)sdTsdGsdTsdGsdGsdTs(5MdC)sascscscsu 118 HAO1A-133394.1 ususgsasasdGsdTsdGsdGsdGsdGsdAsdAsdTsdTsascsasgsa 119 HAO1A-133395.1 cscscsususdTsdGsdTsdAsdTsdTsdGsdAsdAsdGsusgsgsgsg 120 HAO1A-133396.1asasasgsasdAs(5MdC)sdGsdAs(5MdC)sdAs(5MdC)s(5MdC)s(5MdC)sdTsususgsusa121 HAO1 A-133397.1 usasusususdTsdGsdTsdTsdGsdGsdAsdAsdAsdAsgsasascsg122 HAO1 A-133398.1asasgsgsgsdAsdTsdTsdGs(5MdC)sdTsdAsdTsdTsdTsusgsususg 123 HAO1A-133399.1 gscsasasusdGsdAsdAsdAsdTsdAsdAsdAsdAsdGsgsgsasusu 124 HAO1A-133400.1 asasasasgsdTs(5MdC)sdAsdAsdAsdAsdGs(5MdC)sdAsdAsusgsasasa 125HAO1 A-133401.1gsascsascs(5MdC)s(5MdC)sdAsdTsdTsdGsdAsdAsdAsdAsgsuscsasa 126 HAO1A-133402.1 asasasgsgsdTsdTs(5MdC)s(5MdC)sdTsdAsdGsdGsdAs(5MdC)sascscscsa127 HAO1 A-133403.1usususcsusdTsdTs(5MdC)sdTsdAsdAsdAsdAsdGsdGsususcscsu 128 HAO1A-133404.1 usgsasasasdGsdTs(5MdC)s(5MdC)sdAsdTsdTsdTs(5MdC)sdTsususcsusa129 HAO1 A-133405.1usasusasusdTsdTs(5MdC)s(5MdC)sdAsdGsdGsdAsdTsdGsasasasgsu 130 HAO1A-133406.1 usasascsasdGsdTsdTsdAsdAsdTsdAsdTsdAsdTsususcscsa 131 HAO1A-133407.1 gsusususus(5MdC)sdTsdTsdTsdTsdTsdAsdAs(5MdC)sdAsgsususasa 132HAO1 A-133408.1 csascsasusdTsdTsdTs(5MdC)sdAsdAsdTsdGsdTsdTsususcsusu133 HAO1 A-133409.1ascsgsususdGsdTs(5MdC)sdTsdAsdAsdAs(5MdC)sdAs(5MdC)sasusususu 134 HAO1A-133410.1 csasgsgsgsdGsdAsdTsdGsdAs(5MdC)sdGsdTsdTsdGsuscsusasa 135HAO1 A-133411.1csascsususdTsdAsdGs(5MdC)s(5MdC)sdTsdGs(5MdC)s(5MdC)sdAsgsgsgsgsa 136HAO1 A-133412.1asasasgsgsdAsdTsdAs(5MdC)sdAsdGs(5MdC)sdAs(5MdC)sdTsususasgsc 137 HAO1A-133413.1 csasasususdTsdTsdAs(5MdC)sdTsdAsdAsdAsdGsdGsasusascsa 138HAO1 A-133414.1ususgscsusdAs(5MdC)s(5MdC)sdTs(5MdC)s(5MdC)sdAsdAsdTsdTsususascsu 139HAO1 A-133415.1 csascscsusdTsdAsdGsdTsdGsdTsdTsdTsdGs(5MdC)susascscsu140 HAO1 A-133416.1uscsasususdAsdTs(5MdC)sdTsdTsdTsdTs(5MdC)sdAs(5MdC)scsususasg 141 HAO1A-133417.1 asascsasasdTsdGsdAsdGsdAsdTs(5MdC)sdAsdTsdTsasuscsusu 142HAO1 A-133418.1 usascsasgsdGsdTsdTsdAsdAsdTsdAsdAsdAs(5MdC)sasasusgsa143 HAO1 A-133419.1gsusasasas(5MdC)sdAsdGsdAsdAsdTsdAs(5MdC)sdAsdGsgsususasa 144 HAO1A-133420.1 usususasasdAsdGsdAs(5MdC)sdAsdTsdGsdTsdAsdAsascsasgsa 145HAO1 A-133421.1asasgsasas(5MdC)s(5MdC)sdAs(5MdC)sdTsdGsdTsdTsdTsdTsasasasgsa 146 HAO1A-133422.1 csususascsdAsdAsdTsdTsdTsdAsdAsdGsdAsdAscscsascsu 147 HAO1A-133423.1 csusususgsdAsdAs(5MdC)s(5MdC)sdTsdGsdAsdGs(5MdC)sdTsusascsasa148 HAO1 A-133424.1asususascs(5MdC)sdAsdAs(5MdC)sdAs(5MdC)sdTsdTsdTsdGsasascscsu 149 HAO1A-133425.1 usgsusgsasdAsdTs(5MdC)sdAsdGsdGs(5MdC)sdAsdTsdTsascscsasa 150HAO1 A-133426.1 uscsuscsasdAsdAsdGsdTsdTsdGsdTsdGsdAsdAsuscsasgsg 151HAO1 A-133427.1csasgsusgs(5MdC)sdTsdAs(5MdC)s(5MdC)sdTsdTs(5MdC)sdTs(5MdC)sasasasgsu152 HAO1 A-133428.1ususcscsasdAsdTsdTs(5MdC)sdTs(5MdC)sdTs(5MdC)s(5MdC)sdAsgsusgscsu 153HAO1 A-133429.1cscsgscscsdAs(5MdC)s(5MdC)s(5MdC)sdAsdTsdTs(5MdC)s(5MdC)sdAsasususcsu154 HAO1 A-133430.1uscsascscsdAsdAsdTsdTsdAs(5MdC)s(5MdC)sdGs(5MdC)s(5MdC)sascscscsa 155HAO1 A-133431.1 asususcsasdAsdAsdGsdAsdAsdGsdTsdAsdTs(5MdC)sascscsasa156 HAO1 A-133432.1usgsgsasasdAsdTs(5MdC)sdTsdAs(5MdC)sdAsdTsdTs(5MdC)sasasasgsa 157 HAO1A-133433.1 asasgsasusdGsdTsdGsdAsdTsdTsdGsdGsdAsdAsasuscsusa 158 HAO1A-133434.1 ususcsasgsdAs(5MdC)sdAs(5MdC)sdTsdAsdAsdAsdGsdAsusgsusgsa 159HAO1 A-133435.1 csasusususdGsdGsdAsdTsdAsdTsdAsdTsdTs(5MdC)sasgsascsa160 HAO1 A-133436.1csasuscscsdTsdAsdAsdAsdAs(5MdC)sdAsdTsdTsdTsgsgsasusa 161 HAO1A-133437.1asasgsusasdAs(5MdC)sdAsdTsdAs(5MdC)sdAsdTs(5MdC)s(5MdC)susasasasa 162HAO1 A-133438.1usususcsus(5MdC)sdTs(5MdC)sdTsdAsdAsdGsdAsdAsdGsusasascsa 163 HAO1A-133439.1 asasasusgs(5MdC)sdTsdTsdTsdAsdTsdTsdTs(5MdC)sdTscsuscsusa 164

TABLE 4 Unmodified antisense polynucleotides targeting HAO1. SEQ IDSEQ ID Target Oligo Name Oligo transSeq NO: mRNA Target sequence NO:Position HAO1 A-133284.1 GGGAGCAUUUUCACAGGUUA 165 UAACCUGUGAAAAUGCUCCC321 13 HAO1 A-133285.1 AAUUAGCCGGGGGAGCAUUU 166 AAAUGCUCCCCCGGCUAAUU 32223 HAO1 A-133286.1 AUCAUUGAUACAAAUUAGCC 167 GGCUAAUUUGUAUCAAUGAU 323 35HAO1 A-133287.1 GUUGUUCAUAAUCAUUGAUA 168 UAUCAAUGAUUAUGAACAAC 324 45HAO1 A-133288.1 GAUUUAGCAUGUUGUUCAUA 169 UAUGAACAACAUGCUAAAUC 325 55HAO1 A-133289.1 UUGGAAGUACUGAUUUAGCA 170 UGCUAAAUCAGUACUUCCAA 326 66HAO1 A-133290.1 CAUAUAUAGACUUUGGAAGU 171 ACUUCCAAAGUCUAUAUAUG 327 78HAO1 A-133291.1 CUGUAAUAGUCAUAUAUAGA 172 UCUAUAUAUGACUAUUACAG 328 88HAO1 A-133292.1 UUGCCCCAGACCUGUAAUAG 173 CUAUUACAGGUCUGGGGCAA 329 99HAO1 A-133293.1 UUCUUCAUCAUUUGCCCCAG 174 CUGGGGCAAAUGAUGAAGAA 330 110HAO1 A-133294.1 UAUCAGCCAAAGUUUCUUCA 175 UGAAGAAACUUUGGCUGAUA 331 123HAO1 A-133295.1 GCUGCAAUAUUAUCAGCCAA 176 UUGGCUGAUAAUAUUGCAGC 332 133HAO1 A-133296.1 AUCUGGAAAAUGCUGCAAUA 177 UAUUGCAGCAUUUUCCAGAU 333 144HAO1 A-133297.1 GAUACAGCUUCCAUCUGGAA 178 UUCCAGAUGGAAGCUGUAUC 334 156HAO1 A-133298.1 GAGCAUCCUUGGAUACAGCU 179 AGCUGUAUCCAAGGAUGCUC 335 167HAO1 A-133299.1 CAACAUUCCGGAGCAUCCUU 180 AAGGAUGCUCCGGAAUGUUG 336 177HAO1 A-133300.1 GAUCUGUUUCAGCAACAUUC 181 GAAUGUUGCUGAAACAGAUC 337 189HAO1 A-133301.1 AGAAGUCGACAGAUCUGUUU 182 AAACAGAUCUGUCGACUUCU 338 200HAO1 A-133302.1 UGUCCUAAAACAGAAGUCGA 183 UCGACUUCUGUUUUAGGACA 339 211HAO1 A-133303.1 UGCUGACCCUCUGUCCUAAA 184 UUUAGGACAGAGGGUCAGCA 340 222HAO1 A-133304.1 CAUAUUGGCAUGCUGACCCU 185 AGGGUCAGCAUGCCAAUAUG 341 232HAO1 A-133305.1 AGCCCCCACACAUAUUGGCA 186 UGCCAAUAUGUGUGGGGGCU 342 242HAO1 A-133306.1 CUGCAUGGCCGUAGCCCCCA 187 UGGGGGCUACGGCCAUGCAG 343 254HAO1 A-133307.1 GAGCCAUGCGCUGCAUGGCC 188 GGCCAUGCAGCGCAUGGCUC 344 264HAO1 A-133308.1 GCCGUCCACAUGAGCCAUGC 189 GCAUGGCUCAUGUGGACGGC 345 275HAO1 A-133309.1 AGUGGCAAGCUCGCCGUCCA 190 UGGACGGCGAGCUUGCCACU 346 287HAO1 A-133310.1 ACAGGCUCUCACAGUGGCAA 191 UUGCCACUGUGAGAGCCUGU 347 299HAO1 A-133311.1 CAGGGACUGACAGGCUCUCA 192 UGAGAGCCUGUCAGUCCCUG 348 308HAO1 A-133312.1 AUGCCCGUUCCCAGGGACUG 193 CAGUCCCUGGGAACGGGCAU 349 319HAO1 A-133313.1 GAACUCAACAUCAUGCCCGU 194 ACGGGCAUGAUGUUGAGUUC 350 331HAO1 A-133314.1 GAGGUGGCCCAGGAACUCAA 195 UUGAGUUCCUGGGCCACCUC 351 343HAO1 A-133315.1 UCAAUUGAGGAGGUGGCCCA 196 UGGGCCACCUCCUCAAUUGA 352 352HAO1 A-133316.1 CCGCCACUUCUUCAAUUGAG 197 CUCAAUUGAAGAAGUGGCGG 353 363HAO1 A-133317.1 CAGGACCAGCUUCCGCCACU 198 AGUGGCGGAAGCUGGUCCUG 354 375HAO1 A-133318.1 ACGAAGUGCCUCAGGACCAG 199 CUGGUCCUGAGGCACUUCGU 355 386HAO1 A-133319.1 CAGUUGCAGCCAACGAAGUG 200 CACUUCGUUGGCUGCAACUG 356 398HAO1 A-133320.1 UGUAGAUAUACAGUUGCAGC 201 GCUGCAACUGUAUAUCUACA 357 408HAO1 A-133321.1 UCUCGGUCCUUGUAGAUAUA 202 UAUAUCUACAAGGACCGAGA 358 418HAO1 A-133322.1 UUCUUGGUGACUUCUCGGUC 203 GACCGAGAAGUCACCAAGAA 359 430HAO1 A-133323.1 CCGCACUAGCUUCUUGGUGA 204 UCACCAAGAAGCUAGUGCGG 360 440HAO1 A-133324.1 CUUCUCUGCCUGCCGCACUA 205 UAGUGCGGCAGGCAGAGAAG 361 452HAO1 A-133325.1 CUUGUAGCCCAUCUUCUCUG 206 CAGAGAAGAUGGGCUACAAG 362 464HAO1 A-133326.1 AAAUAUGGCCUUGUAGCCCA 207 UGGGCUACAAGGCCAUAUUU 363 473HAO1 A-133327.1 UGUCCACUGUCACAAAUAUG 208 CAUAUUUGUGACAGUGGACA 364 486HAO1 A-133328.1 CAGGUAAGGUGUGUCCACUG 209 CAGUGGACACACCUUACCUG 365 497HAO1 A-133329.1 GACGGUUGCCCAGGUAAGGU 210 ACCUUACCUGGGCAACCGUC 366 507HAO1 A-133330.1 CACAUCAUCCAGACGGUUGC 211 GCAACCGUCUGGAUGAUGUG 367 518HAO1 A-133331.1 AAUCUGUUACGCACAUCAUC 212 GAUGAUGUGCGUAACAGAUU 368 529HAO1 A-133332.1 GCGGCAGUUUGAAUCUGUUA 213 UAACAGAUUCAAACUGCCGC 369 540HAO1 A-133333.1 CUGAGUUGUGGCGGCAGUUU 214 AAACUGCCGCCACAACUCAG 370 550HAO1 A-133334.1 AAAAUUUUUCAUCCUGAGUU 215 AACUCAGGAUGAAAAAUUUU 371 563HAO1 A-133335.1 UACUGGUUUCAAAAUUUUUC 216 GAAAAAUUUUGAAACCAGUA 372 573HAO1 A-133336.1 AAAUGAUAAAGUACUGGUUU 217 AAACCAGUACUUUAUCAUUU 373 584HAO1 A-133337.1 CCUCAGGAGAAAAUGAUAAA 218 UUUAUCAUUUUCUCCUGAGG 374 594HAO1 A-133338.1 UCCAAAAUUUUCCUCAGGAG 219 CUCCUGAGGAAAAUUUUGGA 375 605HAO1 A-133339.1 GUCCACUGUCGUCUCCAAAA 220 UUUUGGAGACGACAGUGGAC 376 618HAO1 A-133340.1 AUAUGCAGCAAGUCCACUGU 221 ACAGUGGACUUGCUGCAUAU 377 629HAO1 A-133341.1 UUUAGCCACAUAUGCAGCAA 222 UUGCUGCAUAUGUGGCUAAA 378 638HAO1 A-133342.1 UGGGUCUAUUGCUUUAGCCA 223 UGGCUAAAGCAAUAGACCCA 379 650HAO1 A-133343.1 AGCUGAUAGAUGGGUCUAUU 224 AAUAGACCCAUCUAUCAGCU 380 660HAO1 A-133344.1 GAUAUCUUCCCAGCUGAUAG 225 CUAUCAGCUGGGAAGAUAUC 381 671HAO1 A-133345.1 CUCAGCCAUUUGAUAUCUUC 226 GAAGAUAUCAAAUGGCUGAG 382 682HAO1 A-133346.1 GAUGUCAGUCUUCUCAGCCA 227 UGGCUGAGAAGACUGACAUC 383 694HAO1 A-133347.1 CAAUUGGCAAUGAUGUCAGU 228 ACUGACAUCAUUGCCAAUUG 384 705HAO1 A-133348.1 CCCUUUGCAACAAUUGGCAA 229 UUGCCAAUUGUUGCAAAGGG 385 715HAO1 A-133349.1 CUCUCAAAAUGCCCUUUGCA 230 UGCAAAGGGCAUUUUGAGAG 386 726HAO1 A-133350.1 GGCAUCAUCACCUCUCAAAA 231 UUUUGAGAGGUGAUGAUGCC 387 737HAO1 A-133351.1 AACAGCCUCCCUGGCAUCAU 232 AUGAUGCCAGGGAGGCUGUU 388 749HAO1 A-133352.1 AAGCCAUGUUUAACAGCCUC 233 GAGGCUGUUAAACAUGGCUU 389 760HAO1 A-133353.1 AAGAUCCCAUUCAAGCCAUG 234 CAUGGCUUGAAUGGGAUCUU 390 772HAO1 A-133354.1 UUCGACACCAAGAUCCCAUU 235 AAUGGGAUCUUGGUGUCGAA 391 781HAO1 A-133355.1 UCGAGCCCCAUGAUUCGACA 236 UGUCGAAUCAUGGGGCUCGA 392 794HAO1 A-133356.1 AUCGAGUUGUCGAGCCCCAU 237 AUGGGGCUCGACAACUCGAU 393 803HAO1 A-133357.1 GGCUGGCACCCCAUCGAGUU 238 AACUCGAUGGGGUGCCAGCC 394 815HAO1 A-133358.1 AACAUCAAUAGUGGCUGGCA 239 UGCCAGCCACUAUUGAUGUU 395 827HAO1 A-133359.1 AUUUCUGGCAGAACAUCAAU 240 AUUGAUGUUCUGCCAGAAAU 396 838HAO1 A-133360.1 AGCCUCCACAAUUUCUGGCA 241 UGCCAGAAAUUGUGGAGGCU 397 848HAO1 A-133361.1 CUUCCCUUCCACAGCCUCCA 242 UGGAGGCUGUGGAAGGGAAG 398 860HAO1 A-133362.1 AAGACUUCCACCUUCCCUUC 243 GAAGGGAAGGUGGAAGUCUU 399 871HAO1 A-133363.1 CCGUCCAGGAAGACUUCCAC 244 GUGGAAGUCUUCCUGGACGG 400 880HAO1 A-133364.1 UCCGCACACCCCCGUCCAGG 245 CCUGGACGGGGGUGUGCGGA 401 891HAO1 A-133365.1 CAUCAGUGCCUUUCCGCACA 246 UGUGCGGAAAGGCACUGAUG 402 903HAO1 A-133366.1 AGCUUUCAGAACAUCAGUGC 247 GCACUGAUGUUCUGAAAGCU 403 914HAO1 A-133367.1 CAAGAGCCAGAGCUUUCAGA 248 UCUGAAAGCUCUGGCUCUUG 404 924HAO1 A-133368.1 ACAGCCUUGGCGCCAAGAGC 249 GCUCUUGGCGCCAAGGCUGU 405 937HAO1 A-133369.1 UCCCCACAAACACAGCCUUG 250 CAAGGCUGUGUUUGUGGGGA 406 948HAO1 A-133370.1 ACGAUUGGUCUCCCCACAAA 251 UUUGUGGGGAGACCAAUCGU 407 958HAO1 A-133371.1 UAAGCCCCAAACGAUUGGUC 252 GACCAAUCGUUUGGGGCUUA 408 968HAO1 A-133372.1 CCCUGGAAAGCUAAGCCCCA 253 UGGGGCUUAGCUUUCCAGGG 409 979HAO1 A-133373.1 CACCUUUCUCCCCCUGGAAA 254 UUUCCAGGGGGAGAAAGGUG 410 990HAO1 A-133374.1 GACAUCUUGAACACCUUUCU 255 AGAAAGGUGUUCAAGAUGUC 411 1001HAO1 A-133375.1 UAGUAUCUCGAGGACAUCUU 256 AAGAUGUCCUCGAGAUACUA 412 1013HAO1 A-133376.1 GAAUUCUUCCUUUAGUAUCU 257 AGAUACUAAAGGAAGAAUUC 413 1025HAO1 A-133377.1 GGCCAACCGGAAUUCUUCCU 258 AGGAAGAAUUCCGGUUGGCC 414 1034HAO1 A-133378.1 CUCAGAGCCAUGGCCAACCG 259 CGGUUGGCCAUGGCUCUGAG 415 1045HAO1 A-133379.1 AUUCUGGCACCCACUCAGAG 260 CUCUGAGUGGGUGCCAGAAU 416 1058HAO1 A-133380.1 AUGACUUUCACAUUCUGGCA 261 UGCCAGAAUGUGAAAGUCAU 417 1069HAO1 A-133381.1 GUCUUGUCGAUGACUUUCAC 262 GUGAAAGUCAUCGACAAGAC 418 1078HAO1 A-133382.1 UUUCCUCACCAAUGUCUUGU 263 ACAAGACAUUGGUGAGGAAA 419 1091HAO1 A-133383.1 CAAAGGAUUUUUCCUCACCA 264 UGGUGAGGAAAAAUCCUUUG 420 1100HAO1 A-133384.1 UUGGAAACGGCCAAAGGAUU 265 AAUCCUUUGGCCGUUUCCAA 421 1111HAO1 A-133385.1 GCACUGUCAGAUCUUGGAAA 266 UUUCCAAGAUCUGACAGUGC 422 1124HAO1 A-133386.1 AAAUAUUGUGCACUGUCAGA 267 UCUGACAGUGCACAAUAUUU 423 1133HAO1 A-133387.1 UACAGAUGGGAAAAUAUUGU 268 ACAAUAUUUUCCCAUCUGUA 424 1144HAO1 A-133388.1 UGAAAAAAAAUAAUACAGAU 269 AUCUGUAUUAUUUUUUUUCA 425 1157HAO1 A-133389.1 UAAUACAUGCUGAAAAAAAA 270 UUUUUUUUCAGCAUGUAUUA 426 1167HAO1 A-133390.1 CUCUUUGUCAAGUAAUACAU 271 AUGUAUUACUUGACAAAGAG 427 1179HAO1 A-133391.1 GCACAGUGUCUCUUUGUCAA 272 UUGACAAAGAGACACUGUGC 428 1188HAO1 A-133392.1 UGGUCACCCUCUGCACAGUG 273 CACUGUGCAGAGGGUGACCA 429 1200HAO1 A-133393.1 UUACAGACUGUGGUCACCCU 274 AGGGUGACCACAGUCUGUAA 430 1210HAO1 A-133394.1 UUGAAGUGGGGAAUUACAGA 275 UCUGUAAUUCCCCACUUCAA 431 1223HAO1 A-133395.1 CCCUUUGUAUUGAAGUGGGG 276 CCCCACUUCAAUACAAAGGG 432 1232HAO1 A-133396.1 AAAGAACGACACCCUUUGUA 277 UACAAAGGGUGUCGUUCUUU 433 1243HAO1 A-133397.1 UAUUUUGUUGGAAAAGAACG 278 CGUUCUUUUCCAACAAAAUA 434 1255HAO1 A-133398.1 AAGGGAUUGCUAUUUUGUUG 279 CAACAAAAUAGCAAUCCCUU 435 1265HAO1 A-133399.1 GCAAUGAAAUAAAAGGGAUU 280 AAUCCCUUUUAUUUCAUUGC 436 1277HAO1 A-133400.1 AAAAGUCAAAAGCAAUGAAA 281 UUUCAUUGCUUUUGACUUUU 437 1288HAO1 A-133401.1 GACACCCAUUGAAAAGUCAA 282 UUGACUUUUCAAUGGGUGUC 438 1299HAO1 A-133402.1 AAAGGUUCCUAGGACACCCA 283 UGGGUGUCCUAGGAACCUUU 439 1311HAO1 A-133403.1 UUUCUUUCUAAAAGGUUCCU 284 AGGAACCUUUUAGAAAGAAA 440 1321HAO1 A-133404.1 UGAAAGUCCAUUUCUUUCUA 285 UAGAAAGAAAUGGACUUUCA 441 1331HAO1 A-133405.1 UAUAUUUCCAGGAUGAAAGU 286 ACUUUCAUCCUGGAAAUAUA 442 1344HAO1 A-133406.1 UAACAGUUAAUAUAUUUCCA 287 UGGAAAUAUAUUAACUGUUA 443 1354HAO1 A-133407.1 GUUUUCUUUUUAACAGUUAA 288 UUAACUGUUAAAAAGAAAAC 444 1364HAO1 A-133408.1 CACAUUUUCAAUGUUUUCUU 289 AAGAAAACAUUGAAAAUGUG 445 1376HAO1 A-133409.1 ACGUUGUCUAAACACAUUUU 290 AAAAUGUGUUUAGACAACGU 446 1388HAO1 A-133410.1 CAGGGGAUGACGUUGUCUAA 291 UUAGACAACGUCAUCCCCUG 447 1397HAO1 A-133411.1 CACUUUAGCCUGCCAGGGGA 292 UCCCCUGGCAGGCUAAAGUG 448 1410HAO1 A-133412.1 AAAGGAUACAGCACUUUAGC 293 GCUAAAGUGCUGUAUCCUUU 449 1421HAO1 A-133413.1 CAAUUUUACUAAAGGAUACA 294 UGUAUCCUUUAGUAAAAUUG 450 1431HAO1 A-133414.1 UUGCUACCUCCAAUUUUACU 295 AGUAAAAUUGGAGGUAGCAA 451 1441HAO1 A-133415.1 CACCUUAGUGUUUGCUACCU 296 AGGUAGCAAACACUAAGGUG 452 1452HAO1 A-133416.1 UCAUUAUCUUUUCACCUUAG 297 CUAAGGUGAAAAGAUAAUGA 453 1464HAO1 A-133417.1 AACAAUGAGAUCAUUAUCUU 298 AAGAUAAUGAUCUCAUUGUU 454 1474HAO1 A-133418.1 UACAGGUUAAUAAACAAUGA 299 UCAUUGUUUAUUAACCUGUA 455 1486HAO1 A-133419.1 GUAAACAGAAUACAGGUUAA 300 UUAACCUGUAUUCUGUUUAC 456 1496HAO1 A-133420.1 UUUAAAGACAUGUAAACAGA 301 UCUGUUUACAUGUCUUUAAA 457 1507HAO1 A-133421.1 AAGAACCACUGUUUUAAAGA 302 UCUUUAAAACAGUGGUUCUU 458 1519HAO1 A-133422.1 CUUACAAUUUAAGAACCACU 303 AGUGGUUCUUAAAUUGUAAG 459 1529HAO1 A-133423.1 CUUUGAACCUGAGCUUACAA 304 UUGUAAGCUCAGGUUCAAAG 460 1542HAO1 A-133424.1 AUUACCAACACUUUGAACCU 305 AGGUUCAAAGUGUUGGUAAU 461 1552HAO1 A-133425.1 UGUGAAUCAGGCAUUACCAA 306 UUGGUAAUGCCUGAUUCACA 462 1564HAO1 A-133426.1 UCUCAAAGUUGUGAAUCAGG 307 CCUGAUUCACAACUUUGAGA 463 1573HAO1 A-133427.1 CAGUGCUACCUUCUCAAAGU 308 ACUUUGAGAAGGUAGCACUG 464 1584HAO1 A-133428.1 UUCCAAUUCUCUCCAGUGCU 309 AGCACUGGAGAGAAUUGGAA 465 1597HAO1 A-133429.1 CCGCCACCCAUUCCAAUUCU 310 AGAAUUGGAAUGGGUGGCGG 466 1607HAO1 A-133430.1 UCACCAAUUACCGCCACCCA 311 UGGGUGGCGGUAAUUGGUGA 467 1617HAO1 A-133431.1 AUUCAAAGAAGUAUCACCAA 312 UUGGUGAUACUUCUUUGAAU 468 1630HAO1 A-133432.1 UGGAAAUCUACAUUCAAAGA 313 UCUUUGAAUGUAGAUUUCCA 469 1641HAO1 A-133433.1 AAGAUGUGAUUGGAAAUCUA 314 UAGAUUUCCAAUCACAUCUU 470 1651HAO1 A-133434.1 UUCAGACACUAAAGAUGUGA 315 UCACAUCUUUAGUGUCUGAA 471 1662HAO1 A-133435.1 CAUUUGGAUAUAUUCAGACA 316 UGUCUGAAUAUAUCCAAAUG 472 1674HAO1 A-133436.1 CAUCCUAAAACAUUUGGAUA 317 UAUCCAAAUGUUUUAGGAUG 473 1684HAO1 A-133437.1 AAGUAACAUACAUCCUAAAA 318 UUUUAGGAUGUAUGUUACUU 474 1694HAO1 A-133438.1 UUUCUCUCUAAGAAGUAACA 319 UGUUACUUCUUAGAGAGAAA 475 1706HAO1 A-133439.1 AAAUGCUUUAUUUCUCUCUA 320 UAGAGAGAAAUAAAGCAUUU 476 1716

TABLE 5 HAO1 Single Dose Screen in Human Hepatocytes. Oligo Name Avg.500 nM SD A-133284.1 51.5 11.4 A-133285.1 54.9 9.0 A-133286.1 49.7 11.0A-133287.1 36.3 4.7 A-133288.1 31.5 5.0 A-133289.1 27.4 2.5 A-133290.157.2 16.1 A-133291.1 56.5 8.5 A-133292.1 32.6 2.2 A-133293.1 42.1 4.1A-133294.1 57.1 32.5 A-133295.1 40.2 12.6 A-133296.1 58.6 28.7A-133297.1 54.2 19.7 A-133298.1 35.2 10.8 A-133299.1 42.2 8.4 A-133300.124.9 4.2 A-133301.1 47.7 5.7 A-133302.1 70.2 10.7 A-133303.1 45.0 3.9A-133304.1 33.9 8.3 A-133305.1 25.2 8.5 A-133306.1 36.1 10.9 A-133307.184.0 24.6 A-133308.1 49.4 8.0 A-133309.1 56.4 3.6 A-133310.1 49.1 6.6A-133311.1 51.4 3.4 A-133312.1 31.3 3.5 A-133313.1 52.5 4.1 A-133314.171.1 8.8 A-133315.1 70.0 22.7 A-133316.1 78.5 35.3 A-133317.1 59.1 21.4A-133318.1 46.2 12.9 A-133319.1 50.0 9.3 A-133320.1 45.1 9.1 A-133321.145.3 3.6 A-133322.1 38.0 4.1 A-133323.1 40.7 5.5 A-133324.1 31.8 8.4A-133325.1 37.1 8.7 A-133326.1 40.9 5.6 A-133327.1 38.4 4.4 A-133328.121.3 3.5 A-133329.1 34.7 4.4 A-133330.1 53.5 10.4 A-133331.1 65.0 10.9A-133332.1 62.3 6.9 A-133333.1 58.8 5.7 A-133334.1 76.6 9.2 A-133335.170.0 5.1 A-133336.1 63.7 4.3 A-133337.1 71.7 3.7 A-133338.1 77.7 18.3A-133339.1 42.0 5.2 A-133340.1 48.0 11.6 A-133341.1 33.7 6.3 A-133342.136.8 10.4 A-133343.1 53.0 11.8 A-133344.1 71.7 9.1 A-133345.1 71.9 22.0A-133346.1 45.5 6.4 A-133347.1 57.7 10.5 A-133348.1 37.2 6.6 A-133349.157.8 10.3 A-133350.1 83.7 17.1 A-133351.1 52.1 6.4 A-133352.1 34.1 4.0A-133353.1 46.3 3.1 A-133354.1 52.0 2.2 A-133355.1 36.4 3.9 A-133356.141.0 7.0 A-133357.1 44.9 5.0 A-133358.1 69.8 3.8 A-133359.1 53.0 3.9A-133360.1 35.0 2.3 A-133361.1 34.2 2.4 A-133362.1 69.5 4.5 A-133363.149.3 4.0 A-133364.1 20.0 3.6 A-133365.1 36.4 4.8 A-133366.1 27.3 2.2A-133367.1 35.8 6.7 A-133368.1 26.1 3.9 A-133369.1 24.1 5.6 A-133370.146.7 6.2 A-133371.1 22.9 4.2 A-133372.1 20.9 4.7 A-133373.1 39.4 4.2A-133374.1 85.2 8.4 A-133375.1 49.3 3.8 A-133376.1 83.1 9.1 A-133377.138.6 5.0 A-133378.1 32.4 6.1 A-133379.1 48.3 15.0 A-133380.1 44.5 14.6A-133381.1 41.8 14.5 A-133382.1 62.5 23.6 A-133383.1 62.8 19.5A-133384.1 36.0 6.8 A-133385.1 34.3 5.6 A-133386.1 44.9 11.8 A-133387.160.1 11.0 A-133388.1 71.6 14.4 A-133389.1 67.0 13.3 A-133390.1 38.7 8.9A-133391.1 20.2 4.6 A-133392.1 18.0 3.3 A-133393.1 15.6 2.3 A-133394.198.3 10.1 A-133395.1 39.0 3.2 A-133396.1 23.4 7.0 A-133397.1 80.4 7.9A-133398.1 64.8 9.7 A-133399.1 86.8 17.9 A-133400.1 63.1 6.9 A-133401.124.0 5.6 A-133402.1 15.8 1.0 A-133403.1 53.5 6.7 A-133404.1 50.3 7.4A-133405.1 47.0 6.8 A-133406.1 72.1 11.9 A-133407.1 57.1 13.2 A-133408.160.2 11.7 A-133409.1 42.2 9.4 A-133410.1 79.8 15.8 A-133411.1 77.7 15.1A-133412.1 28.6 5.5 A-133413.1 96.3 8.5 A-133414.1 65.0 7.8 A-133415.128.3 2.7 A-133416.1 62.7 12.2 A-133417.1 71.1 6.6 A-133418.1 74.8 14.2A-133419.1 49.4 6.9 A-133420.1 82.6 19.7 A-133421.1 38.0 7.2 A-133422.178.3 13.6 A-133423.1 47.7 8.4 A-133424.1 41.9 9.8 A-133425.1 14.0 4.0A-133426.1 25.3 6.3 A-133427.1 41.8 7.3 A-133428.1 42.6 6.7 A-133429.162.0 3.1 A-133430.1 44.3 4.0 A-133431.1 78.5 5.9 A-133432.1 62.7 9.5A-133433.1 51.0 9.8 A-133434.1 35.3 2.8 A-133435.1 59.0 1.9 A-133436.188.7 11.0 A-133437.1 81.0 11.4 A-133438.1 55.3 3.7 A-133439.1 37.6 6.4

Example 3: In Vivo HAO1 Silencing in a Mouse Expressing hHAO1

Experiments are performed to assess hHAO1 target knockdown in a mouseexpressing hHAO1 from an AAV8 expression vector. Briefly, mice areinfected with 1×10¹¹ AAV8 particles containing an expression constructfor human AAV under the control of a constitutive promoter. Two weeksafter infection with the AAV8 virus, mice are administered single dosesof various amounts of a polynucleotide agent targeting HAO1 or PBS as acontrol. The level of expression of hHAO1 is assessed by qPCR and ELISAof liver tissue at predetermined time points after administration of thepolynucleotide agent. Polynucleotide agents with the most potent hHAO1knockdown activity are selected for further testing.

Example 4. Amelioration of Hyperoxaluria In Vivo by HAO1 Silencing

Mouse model of PH1 that includes loss of function mutations in the AGXTgene or a mouse model of PH2 that includes a knockout of the GRHPR gene(Salido E C, et al. (2006) PNAS 103(48): 18249-18254 and Knight J, etal. (2012) Am. J. Physiol. Renal Physiol. 302: F688-F693) are used forin vivo experiments. The mice are administered an appropriate dose of apolynucleotide agent provided herein targeting HAO1. A second subset ofmice are left untreated as a control. Dosing is repeated. The mice aresubjected to urinary analysis, sediment analysis and analysis for thedevelopment of urinary bladder stones.

We claim:
 1. A polynucleotide agent for inhibiting expression ofhydroxyacid oxidase (HAO1), wherein the agent comprises about 4 to about50 contiguous nucleotides, wherein at least one of the contiguousnucleotides is a modified nucleotide, and wherein the nucleotidesequence of the agent is about 80% complementary over its entire lengthto the equivalent region of the nucleotide sequence of any one of SEQ IDNOs:1-4.
 2. The agent of claim 1, wherein the equivalent region is anyone of the regions of SEQ ID NO:1 targeted by the agents provided inTable 3 or Table
 4. 3. A polynucleotide agent for inhibiting expressionof hydroxyacid oxidase (HAO1), wherein the agent comprises at least 8contiguous nucleotides differing by no more than 3 nucleotides from anyone of the nucleotide sequences listed in Tables 3 and 4, and whereinthe agent is about 8 to about 50 nucleotides in length.
 4. The agent ofclaim 1, wherein substantially all of the nucleotides of the antisensepolynucleotide agent are modified nucleotides; or wherein all of thenucleotides of the antisense polynucleotide agent are modifiednucleotides.
 5. The agent of claim 1, which is 10 to 40 nucleotides inlength; 10 to 30 nucleotides in length; 18 to 30 nucleotides in length;10 to 24 nucleotides in length; 18 to 24 nucleotides in length; or 20nucleotides in length.
 6. The agent of claim 1, wherein the modifiednucleotide comprises a modified sugar moiety selected from the groupconsisting of: a 2′-O-methoxyethyl modified sugar moiety, a 2′-O-alkylmodified sugar moiety, and a bicyclic sugar moiety.
 7. The agent ofclaim 1, wherein the modified nucleotide is a 5-methylcytosine.
 8. Theagent of claim 1, wherein the modified nucleotide comprises a modifiedinternucleoside linkage.
 9. The agent of claim 1, comprising a pluralityof 2′-deoxynucleotides flanked on each side by at least one nucleotidehaving a modified sugar moiety.
 10. The agent of claim 9, wherein theagent is a gapmer comprising a gap segment comprised of linked2′-deoxynucleotides positioned between a 5′ and a 3′ wing segment.
 11. Apolynucleotide agent for inhibiting hydroxyacid oxidase (HAO1)expression, comprising a gap segment consisting of linkeddeoxynucleotides; a 5′-wing segment consisting of linked nucleotides; a3′-wing segment consisting of linked nucleotides; wherein the gapsegment is positioned between the 5′-wing segment and the 3′-wingsegment and wherein each nucleotide of each wing segment comprises amodified sugar.
 12. The agent of claim 11, wherein the gap segment isten 2′-deoxynucleotides in length and each of the wing segments is fivenucleotides in length.
 13. The agent of claim 1, wherein the agentfurther comprises a ligand at the 3′-terminus.
 14. The agent of claim13, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.15. A pharmaceutical composition for inhibiting expression of anhydroxyacid oxidase (HAO1) gene comprising the agent of claim
 1. 16. Apharmaceutical composition comprising the agent of claim 1, and a lipidformulation.
 17. A method of inhibiting hydroxyacid oxidase (HAO1)expression in a cell, the method comprising: (a) contacting the cellwith the agent of claim 1 or a pharmaceutical composition of claim 15;and (b) maintaining the cell produced in step (a) for a time sufficientto obtain antisense inhibition of an HAO1 gene, thereby inhibitingexpression of the HAO1 gene in the cell.
 18. The method of claim 17,wherein the cell is within a subject.
 19. The method of claim 18,wherein the subject is a human.
 20. A method of treating a subjecthaving a disease or disorder that would benefit from reduction inhydroxyacid oxidase (HAO1) expression, the method comprisingadministering to the subject a therapeutically effective amount of theagent of claim 1 or a pharmaceutical composition of claim 15, therebytreating the subject.
 21. A method of preventing at least one symptom ina subject having a disease or disorder that would benefit from reductionin hydroxyacid oxidase (HAO1) expression, the method comprisingadministering to the subject a prophylactically effective amount ofclaim 1 or a pharmaceutical composition of claim 15, thereby preventingat least one symptom in the subject having a disorder that would benefitfrom reduction in HAO1 expression.
 22. The method of claim 20 or 21,wherein the disorder is an hydroxyacid oxidase (HAO1)-associateddisease.
 23. The method of claim 22, wherein the HAO1-associated diseaseis selected from the group consisting of primary hyperoxaluria andsecondary hyperoxaluria.